Methods and compositions for RNA interference

ABSTRACT

The present invention provides methods for attenuating gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA), such as a hairpin RNA. The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/350,798, filed on Jan. 24, 2003, which is a continuation-in-part ofU.S. application Ser. No. 10/055,797, filed on Jan. 22, 2002, which is acontinuation-in-part of International Application No. PCT/US01/08435,filed on Mar. 16, 2001, which claims the benefit of priority from U.S.Provisional Application Nos. 60/189,739, filed on Mar. 16, 2000, and60/243,097, filed on Oct. 24, 2000. U.S. application Ser. No. 10/350,798is also a continuation-in-part of U.S. application Ser. No. 09/866,557,filed on May 24, 2001, which is a continuation-in-part of InternationalApplication No. PCT/US01/08435, filed on Mar. 16, 2001, which claims thebenefit of priority from U.S. Provisional Application Nos. 60/189,739,filed on Mar. 16, 2000, and 60/243,097, filed on Oct. 24, 2000. U.S.application Ser. No. 10/350,798 is also a continuation-in-part of U.S.application Ser. No. 09/858,862, filed on May 16, 2001, which is acontinuation-in-part of International Application No. PCT/US01/08435,filed on Mar. 16, 2001, which claims the benefit of priority from U.S.Provisional Application Nos. 60/189,739, filed on Mar. 16, 2000, and60/243,097, filed on Oct. 24, 2000. The specifications of suchapplications are incorporated by reference herein. InternationalApplication PCT/US01/08435 was published under PCT Article 21(2) inEnglish.

GOVERNMENT SUPPORT

Work described herein was supported by National Institutes of HealthGrant R01-GM62534. The United States Government may have certain rightsin the invention.

BACKGROUND OF THE INVENTION

“RNA interference”, “post-transcriptional gene silencing”,“quelling”—these different names describe similar effects that resultfrom the overexpression or misexpression of transgenes, or from thedeliberate introduction of double-stranded RNA into cells (reviewed inFire, Trends Genet 15: 358-363, 1999; Sharp, Genes Dev 13: 139-141,1999; Hunter, Curr Biol 9: R440-R442, 1999; Baulcombe, Curr Biol 9:R599-R601, 1999; Vaucheret et al., Plant J 16: 651-659, 1998). Theinjection of double-stranded RNA into the nematode Caenorhabditiselegans, for example, acts systemically to cause thepost-transcriptional depletion of the homologous endogenous RNA (Fire etal., Nature 391: 806-811, 1998; and Montgomery et al., PNAS 95:15502-15507, 1998). RNA interference, commonly referred to as RNAi,offers a way of specifically and potently inactivating a cloned gene,and is proving a powerful tool for investigating gene function. Althoughthe phenomenon is interesting in its own right; the mechanism has beenrather mysterious, but recent research—for example that recentlyreported by Smardon et al., Curr Biol 10: 169-178, 2000—is beginning toshed light on the nature and evolution of the biological processes thatunderlie RNAi.

RNAi was discovered when researchers attempting to use the antisense RNAapproach to inactivate a C. elegans gene found that injection ofsense-strand RNA was actually as effective as the antisense RNA atinhibiting gene function (Guo et al., Cell 81: 611-620, 1995). Furtherinvestigation revealed that the active agent was modest amounts ofdouble-stranded RNA that contaminate in vitro RNA preparations.Researchers quickly determined the ‘rules’ and effects of RNAi whichhave become the paradigm for thinking about the mechanism which mediatesthis affect. Exon sequences are required, whereas introns and promotersequences, while ineffective, do not appear to compromise RNAi (thoughthere may be gene-specific exceptions to this rule). RNAi actssystemically—injection into one tissue inhibits gene function in cellsthroughout the animal. The results of a variety of experiments, in C.elegans and other organisms, indicate that RNAi acts to destabilizecellular RNA after RNA processing.

The potency of RNAi inspired Timmons and Fire (Nature 395: 854, 1998) todo a simple experiment that produced an astonishing result. They fed tonematodes bacteria that had been engineered to express double-strandedRNA corresponding to the C. elegans unc-22 gene. Amazingly, thesenematodes developed a phenotype similar to that of unc-22 mutants thatwas dependent on their food source. The ability to conditionally exposelarge numbers of nematodes to gene-specific double-stranded RNA formedthe basis for a very powerful screen to select for RNAi-defective C.elegans mutants and then to identify the corresponding genes.

Double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous invitro contexts including Drosophila, Caenorhabditis elegans, planaria,hydra, trypanosomes, fungi and plants. However, the ability torecapitulate this phenomenon in higher eukaryotes, particularlymammalian cells, has not been accomplished in the art. Nor has the priorart demonstrated that this phenomena can be observed in culturedeukaryotic cells. Additionally, the ‘rules’ established by the prior arthave taught that RNAi requires exon sequences, and thus constructsconsisting of intronic or promoter sequences were not believed to beeffective reagents in mediating RNAi. The present invention aims toaddress each of these deficiencies in the prior art and providesevidence both that RNAi can be observed in cultured eukaryotic cells andthat RNAi constructs consisting of non-exon sequences can effectivelyrepress gene expression.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method for attenuatingexpression of a target gene in cultured cells, comprising introducingdouble stranded RNA (dsRNA) into the cells in an amount sufficient toattenuate expression of the target gene, wherein the dsRNA comprises anucleotide sequence that hybridizes under stringent conditions to anucleotide sequence of the target gene.

Another aspect of the present invention provides a method forattenuating expression of a target gene in a mammalian cell, comprising:(i) activating one or both of a Dicer activity or an Argonaut activityin the cell, and (ii) introducing into the cell a double stranded RNA(dsRNA) in an amount sufficient to attenuate expression of the targetgene, wherein the dsRNA comprises a nucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence of the target gene.

In certain embodiments, the cell is suspended in culture; while in otherembodiments the cell is in a whole animal, such as a non-human mammal.

In certain preferred embodiments, the cell is engineered with (i) arecombinant gene encoding a Dicer activity, (ii) a recombinant geneencoding an Argonaut activity, or (iii) both. For instance, therecombinant gene may encode, for a example, a protein which includes anamino acid sequence at least 50 percent identical to SEQ ID NO: 2 or 4;or be defined by a coding sequence which hybridizes under washconditions of 2×SSC at 22° C. to SEQ ID NO: 1 or 3. In certainembodiments, the recombinant gene may encode, for a example, a proteinwhich includes an amino acid sequence at least 50 percent identical tothe Argonaut sequence shown in FIG. 24. In certain embodiments, therecombinant gene may encode a protein which includes an amino acidsequence at least 60%, 70%, 80%, 85%, 90%, or 95% identical to SEQ IDNO: 2 or 4. In certain embodiments, the recombinant gene may be definedby a coding sequence which hybridizes under stringent conditions,including a wash step selected from 0.2-2.0×SSC at from 50° C.-65° C.,to SEQ ID NO: 1 or 3.

In certain embodiments, rather than use a heterologous expressionconstruct(s), an endogenous Dicer gene or Argonaut gene can beactivated, e.g., by gene activation technology, expression of activatedtranscription factors or other signal transduction protein(s), whichinduces expression of the gene, or by treatment with an endogenousfactor which upregulates the level of expression of the protein orinhibits the degradation of the protein.

In certain preferred embodiments, the target gene is an endogenous geneof the cell. In other embodiments, the target gene is a heterologousgene relative to the genome of the cell, such as a pathogen gene, e.g.,a viral gene.

In certain embodiments, the cell is treated with an agent that inhibitsprotein kinase RNA-activated (PKR) apoptosis, such as by treatment withagents which inhibit expression of PKR, cause its destruction, and/orinhibit the kinase activity of PKR.

In certain preferred embodiments, the cell is a primate cell, such as ahuman cell.

In certain preferred embodiments, the length of the dsRNA is at least20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNAproducts produced by Dicer-dependent cleavage. In certain embodiments,the dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases. Incertain embodiments, the dsRNA construct is 400-800 bases in length.

In certain preferred embodiments, expression of the target gene isattenuated by at least 5 fold, and more preferably at least 10, 20 oreven 50 fold, e.g., relative to the untreated cell or a cell treatedwith a dsRNA construct which does not correspond to the target gene.

Yet another aspect of the present invention provides a method forattenuating expression of a target gene in cultured cells, comprisingintroducing an expression vector having a “coding sequence” which, whentranscribed, produces double stranded RNA (dsRNA) in the cell in anamount sufficient to attenuate expression of the target gene, whereinthe dsRNA comprises a nucleotide sequence that hybridizes understringent conditions to a nucleotide sequence of the target gene. Incertain embodiments, the vector includes a single coding sequence forthe dsRNA which is operably linked to (two) transcriptional regulatorysequences which cause transcription in both directions to formcomplementary transcripts of the coding sequence. In other embodiments,the vector includes two coding sequences which, respectively, give riseto the two complementary sequences which form the dsRNA when annealed.In still other embodiments, the vector includes a coding sequence whichforms a hairpin. In certain embodiments, the vectors are episomal, e.g.,and transfection is transient. In other embodiments, the vectors arechromosomally integrated, e.g., to produce a stably transfected cellline. Preferred vectors for forming such stable cell lines are describedin U.S. Pat. No. 6,025,192 and PCT publication WO 98/12339, which areincorporated by reference herein.

Another aspect of the present invention provides a method forattenuating expression of a target gene in cultured cells, comprisingintroducing an expression vector having a “non-coding sequence” which,when transcribed, produces double stranded RNA (dsRNA) in the cell in anamount sufficient to attenuate expression of the target gene. Thenon-coding sequence may include intronic or promoter sequence of thetarget gene of interest, and the dsRNA comprises a nucleotide sequencethat hybridizes under stringent conditions to a nucleotide sequence ofthe promoter or intron of the target gene. In certain embodiments, thevector includes a single sequence for the dsRNA which is operably linkedto (two) transcriptional regulatory sequences which cause transcriptionin both directions to form complementary transcripts of the sequence. Inother embodiments, the vector includes two sequences which,respectively, give rise to the two complementary sequences which formthe dsRNA when annealed. In still other embodiments, the vector includesa coding sequence which forms a hairpin. In certain embodiments, thevectors are episomal, e.g., and transfection is transient. In otherembodiments, the vectors are chromosomally integrated, e.g., to producea stably transfected cell line. Preferred vectors for forming suchstable cell lines are described in U.S. Pat. No. 6,025,192 and PCTpublication WO 98/12339, which are incorporated by reference herein.

Another aspect the present invention provides a double stranded (ds) RNAfor inhibiting expression of a mammalian gene. The dsRNA comprises afirst nucleotide sequence that hybridizes under stringent conditions,including a wash step of 0.2×SSC at 65° C., to a nucleotide sequence ofat least one mammalian gene and a second nucleotide sequence which iscomplementary to the first nucleotide sequence.

In one embodiment, the first nucleotide sequence of said double-strandedRNA is at least 20, 21, 22, 25, 50, 100, 200, 300, 400, 500, 800nucleotides in length.

In another embodiment, the first nucleotide sequence of saiddouble-stranded RNA is identical to at least one mammalian gene. Inanother embodiment, the first nucleotide sequence of saiddouble-stranded RNA is identical to one mammalian gene. In yet anotherembodiment, the first nucleotide sequence of said double-stranded RNAhybridizes under stringent conditions to at least one human gene. Instill another embodiment, the first nucleotide sequence of saiddouble-stranded RNA is identical to at least one human gene. In stillanother embodiment, the first nucleotide sequence of saiddouble-stranded RNA is identical to one human gene.

The double-stranded RNA may be an siRNA or a hairpin, and may beexpressed transiently or stably. In one embodiment, the double-strandedRNA is a hairpin comprising a first nucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence of at least onemammalian gene, and a second nucleotide sequence which is acomplementary inverted repeat of said first nucleotide sequence andhybridizes to said first nucleotide sequence to form a hairpinstructure.

The first nucleotide sequence of said double-stranded RNA can hybridizeto either coding or non-coding sequence of at least one mammalian gene.In one embodiment, the first nucleotide sequence of said double-strandedRNA hybridizes to a coding sequence of at least one mammalian gene. Inanother embodiment, the first nucleotide sequence of saiddouble-stranded RNA hybridizes to a coding sequence of at least onehuman gene. In another embodiment, the first nucleotide sequence of saiddouble-stranded RNA is identical to a coding sequence of at least onemammalian gene. In still another embodiment, the first nucleotidesequence of said double-stranded RNA is identical to a coding sequenceof at least one human gene.

In another embodiment, the first nucleotide sequence of saiddouble-stranded RNA hybridizes to a non-coding sequence of at least onemammalian gene. In another embodiment, the first nucleotide sequence ofsaid double-stranded RNA hybridizes to a non-coding sequence of at leastone human gene. In another embodiment, the first nucleotide sequence ofsaid double-stranded RNA is identical to a non-coding sequence of atleast one mammalian gene. In still another embodiment, the firstnucleotide sequence of said double-stranded RNA is identical to anon-coding sequence of at least one human gene. In any of the foregoingembodiments, the non-coding sequence may be a non-transcribed sequence.

Still another aspect of the present invention provides an assay foridentifying nucleic acid sequences, either coding or non-codingsequences, responsible for conferring a particular phenotype in a cell,comprising: (i) constructing a variegated library of nucleic acidsequences from a cell in an orientation relative to a promoter toproduce double stranded DNA; (ii) introducing the variegated dsRNAlibrary into a culture of target cells; (iii) identifying members of thelibrary which confer a particular phenotype on the cell, and identifyingthe sequence from a cell which correspond, such as being identical orhomologous, to the library member.

Yet another aspect of the present invention provides a method ofconducting a drug discovery business comprising: (i) identifying, by thesubject assay, a target gene which provides a phenotypically desirableresponse when inhibited by RNAi; (ii) identifying agents by theirability to inhibit expression of the target gene or the activity of anexpression product of the target gene; (iii) conducting therapeuticprofiling of agents identified in step (b), or further analogs thereof,for efficacy and toxicity in animals; and (iv) formulating apharmaceutical preparation including one or more agents identified instep (iii) as having an acceptable therapeutic profile.

The method may include an additional step of establishing a distributionsystem for distributing the pharmaceutical preparation for sale, and mayoptionally include establishing a sales group for marketing thepharmaceutical preparation.

Another aspect of the present invention provides a method of conductinga target discovery business comprising: (i) identifying, by the subjectassay, a target gene which provides a phenotypically desirable responsewhen inhibited by RNAi; (ii) (optionally) conducting therapeuticprofiling of the target gene for efficacy and toxicity in animals; and(iii) licensing, to a third party, the rights for further drugdevelopment of inhibitors of the target gene.

Another aspect of the invention provides a method for inhibiting RNAi byinhibiting the expression or activity of an RNAi enzyme. Thus, thesubject method may include inhibiting the activity of Dicer and/or the22-mer RNA.

Still another aspect relates to a method for altering the specificity ofan RNAi by modifying the sequence of the RNA component of the RNAienzyme.

In another aspect, gene expression in an undifferentiated stem cell, orthe differentiated progeny thereof, is altered by introducing dsRNA ofthe present invention. In one embodiment, the stem cells are embryonicstem cells. Preferably, the embryonic stem cells are derived frommammals, more preferably from non-human primates, and most preferablyfrom humans.

The embryonic stem cells may be isolated by methods known to one ofskill in the art from the inner cell mass (ICM) of blastocyst stageembryos. In one embodiment the embryonic stem cells are obtained frompreviously established cell lines. In a second embodiment, the embryonicstem cells are derived de novo by standard methods.

In another aspect, the embryonic stem cells are the result of nucleartransfer. The donor nuclei are obtained from any adult, fetal, orembryonic tissue by methods well known in the art. In one embodiment,the donor nuclei is transferred to a recipient oocyte which hadpreviously been modified. In one embodiment, the oocyte is modifiedusing one or more dsRNAs. Exemplary modifications of the recipientoocyte include any changes in gene or protein expression that prevent anembryo derived from said modified oocyte from successfully implanting inthe uterine wall. Since implantation in the uterine wall is essentialfor fertilized mammalian embryos to progress from beyond the blastocyststage, embryos made from such modified oocytes could not give rise toviable organisms. Non-limiting examples of such modifications includethose that decrease or eliminate expression of cell surface receptors(i.e., integrins) required for the recognition between the blastocystand the uterine wall, modifications that decrease or eliminateexpression of proteases (i.e., collagenase, stromelysin, and plasminogenactivator) required to digest matrix in the uterine lining and thusallow proper implantation, and modifications that decrease or eliminateexpression of proteases (i.e., trypsin) necessary for the blastocyst tohatch from the zona pellucida. Such hatching is required forimplantation.

In another embodiment, embryonic stem cells, embryonic stem cellsobtained from fertilization of modified oocytes, or the differentiatedprogeny thereof, can be modified or further modified with one or moredsRNAs. In a preferred embodiment, the modification decreases oreliminates MHC expression. Cells modified in this way will be toleratedby the recipient, thus avoiding complications arising from graftrejection. Such modified cells are suitable for transplantation into arelated or unrelated patient to treat a condition characterized by celldamage or cell loss.

In another aspect of the invention, the undifferentiated stem cell is anadult stem cell. Exemplary adult stem cells include, but are not limitedto, hematopoietic stem cells, mesenchymal stem cells, cardiac stemcells, pancreatic stem cells, and neural stem cells. Exemplary adultstem cells include any stem cell capable of forming differentiatedectodermal, mesodermal, or endodermal derivatives. Non-limiting examplesof differentiated cell types which arise from adult stem cells include:blood, skeletal muscle, myocardium, endocardium, pericardium, bone,cartilage, tendon, ligament, connective tissue, adipose tissue, liver,pancreas, skin, neural tissue, lung, small intestine, large intestine,gall bladder, rectum, anus, bladder, female or male reproductive tract,genitals, and the linings of the body cavity.

In one embodiment, an undifferentiated adult stem cell, or thedifferentiated progeny thereof, is altered with one or more dsRNAs todecrease or eliminate MHC expression. Cells modified in this way will betolerated by the recipient, thus avoiding complications arising fromgraft rejection. Such modified cells are suitable for transplantationinto a related or unrelated patient to treat a condition characterizedby cell damage or cell loss.

In another aspect of the invention, an embryonic stem cell, anundifferentiated adult stem cell, or the differentiated progeny ofeither an embryonic or adult stem cell is altered with one or more dsRNAto decrease or eliminate expression of genes required for HIV infection.In a preferred embodiment, the stem cell is one capable of giving riseto hematopoietic cells. Modified cells with hematopoietic potential canbe transplanted into a patient as a preventative therapy or treatmentfor HIV or AIDS.

Another aspect of the invention relates to purified or semi-purifiedpreparations of the RNAi enzyme or components thereof. In certainembodiments, the preparations are used for identifying compounds,especially small organic molecules, which inhibit or potentiate the RNAiactivity. Small molecule inhibitors, for example, can be used to inhibitdsRNA responses in cells which are purposefully being transfected with avirus which produces double stranded RNA.

The dsRNA construct may comprise one or more strands of polymerizedribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. The double-strandedstructure may be formed by a single self-complementary RNA strand or twocomplementary RNA strands. RNA duplex formation may be initiated eitherinside or outside the cell. The dsRNA construct may be introduced in anamount which allows delivery of at least one copy per cell. Higher dosesof double-stranded material may yield more effective inhibition.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. In certain embodiments, dsRNA constructs containing anucleotide sequences identical to a portion of the target gene arepreferred for inhibition. RNA sequences with insertions, deletions, andsingle point mutations relative to the target sequence (i.e., RNAsequences similar to the target sequence) have also been found to beeffective for inhibition. Thus, sequence identity may be optimized byalignment algorithms known in the art and calculating the percentdifference between the nucleotide sequences. Alternatively, the duplexregion of the RNA may be defined functionally as a nucleotide sequencethat is capable of hybridizing with a portion of the target genetranscript. In another embodiment, dsRNA constructs containingnucleotide sequences identical to a non-coding portion of the targetgene are preferred for inhibition. Exemplary non-coding regions includeintrons and the promoter region. Sequences with insertions, deletions,and single point mutations relative to the target non-coding sequencemay also be used.

Yet another aspect of the invention pertains to transgenic non-humanmammals which include a transgene encoding a dsRNA construct, whereinthe dsRNA is identical or similar to either the coding or non-codingsequence of the target gene, preferably which is stably integrated intothe genome of cells in which it occurs. The animals can be derived byoocyte microinjection, for example, in which case all of the nucleatedcells of the animal will include the transgene, or can be derived usingembryonic stem (ES) cells which have been transfected with thetransgene, in which case the animal is a chimera and only a portion ofits nucleated cells will include the transgene. In certain instances,the sequence-independent dsRNA response, e.g., the PKR response, is alsoinhibited in those cells including the transgene.

In still other embodiments, dsRNA itself can be introduced into an EScell in order to effect gene silencing, and that phenotype will becarried for at least several rounds of division, e.g., into the progenyof that cell.

Another aspect of the invention provides a method for attenuatingexpression of a target gene in mammalian cells, comprising introducinginto the mammalian cells a single-stranded hairpin ribonucleic acid(shRNA) comprising self complementary sequences of 19 to 100 nucleotidesthat form a duplex region, which self complementary sequences hybridizeunder intracellular conditions to a target gene, wherein said hairpinRNA: (i) is a substrate for cleavage by a RNaseIII enzyme to produce adouble-stranded RNA product, (ii) does not produce a generalsequence-independent killing of the mammalian cells, and (iii) reducesexpression of said target gene in a manner dependent on the sequence ofsaid complementary regions. Preferably, the shRNA comprises a 3′overhang of about 1-4 nucleotides.

A related aspect of the invention provides a method for attenuatingexpression of a target gene in mammalian cells, comprising introducinginto the mammalian cells a single-stranded hairpin ribonucleic acid(shRNA) comprising self complementary sequences of 19 to 100 nucleotidesthat form a duplex region, which self complementary sequences hybridizeunder intracellular conditions to a target gene, wherein said hairpinRNA: (i) is cleaved in the mammalian cells to produce an RNA guidesequence that enters an Argonaut-containing complex, (ii) does notproduce a general sequence-independent killing of the mammalian cells,and (iii) reduces expression of said target gene in a manner dependenton the sequence of said complementary regions. Preferably, the shRNAcomprises a 3′ overhang of about 1-4 nucleotides.

Yet another related aspect of the invention provides a method forattenuating expression of one or more target genes in mammalian cells,comprising introducing into the mammalian cells a variegated library ofsingle-stranded hairpin ribonucleic acid (shRNA) species, each shRNAspecies comprising self complementary sequences of 19 to 100 nucleotidesthat form duplex regions and which hybridize under intracellularconditions to a target gene, wherein each of said hairpin RNA species:(i) is a substrate for cleavage by a RNaseIII enzyme to produce adouble-stranded RNA product, (ii) does not produce a generalsequence-independent killing of the mammalian cells, and (iii) ifcomplementary to a target sequence, reduces expression of said targetgene in a manner dependent on the sequence of said complementaryregions. Preferably, the shRNA comprises a 3′ overhang of about 1-4nucleotides.

In one embodiment, the shRNA comprises a 3′ overhang of 2 nucleotides.

In one embodiment, the shRNA comprises self-complementary sequences of25 to 29 nucleotides that form duplex regions.

In one embodiment, the self-complementary sequences are 29 nucleotidesin length.

In one embodiment, the shRNA is transfected or microinjected into saidmammalian cells.

In one embodiment, the shRNA is a transcriptional product that istranscribed from an expression construct introduced into said mammaliancells, which expression construct comprises a coding sequence fortranscribing said shRNA, operably linked to one or more transcriptionalregulatory sequences. The transcriptional regulatory sequences mayinclude a promoter for an RNA polymerase, such as a cellular RNApolymerase.

In one embodiment, the promoter is a U6 promoter, a T7 promoter, a T3promoter, or an SP6 promoter.

In one embodiment, the transcriptional regulatory sequences includes aninducible promoter.

In one embodiment, the mammalian cells are stably transfected with saidexpression construct.

In one embodiment, the mammalian cells are primate cells, such as humancells.

In one embodiment, the shRNA is introduced into the mammalian cells incell culture or in an animal.

In one embodiment, the expression of the target is attenuated by atleast 33 percent relative expression in cells not treated said hairpinRNA.

In one embodiment, the target gene is an endogenous gene or aheterologous gene relative to the genome of the mammalian cell.

In one embodiment, the self complementary sequences hybridize underintracellular conditions to a non-coding sequence of the target geneselected from a promoter sequence, an enhancer sequence, or an intronicsequence.

In one embodiment, the shRNA includes one or more modifications tophosphate-sugar backbone or nucleosides residues.

In one embodiment, the variegated library of shRNA species are arrayed asolid substrate.

In one embodiment, the method includes the further step of identifyingshRNA species of said variegated library which produce a detectedphenotype in said mammalian cells.

In one embodiment, the shRNA is a chemically synthesized product or anin vitro transcription product.

Another aspect of the invention provides a method of enhancing thepotency/activity of an RNAi therapeutic for a mammalian patient, saidRNAi therapeutic comprising an siRNA of 19-22 paired polynucleotides,the method comprising replacing said siRNA with a single-strandedhairpin RNA (shRNA) of claim 1 or 2, wherein said duplex regioncomprises the same 19-22 paired polynucleotides of said siRNA.

In one embodiment, the shRNA comprises a 3′ overhang of 2 nucleotides.

In one embodiment, the half-maximum inhibition by said RNAi therapeuticis achieved by a concentration of said shRNA at least about 20% lowerthan that of said siRNA.

In one embodiment, the half-maximum inhibition by said RNAi therapeuticis achieved by a concentration of said shRNA at least about 100% lowerthan that of said siRNA.

In one embodiment, the end-point inhibition by said shRNA is at leastabout 40% higher than that of said siRNA.

In one embodiment, the end-point inhibition by said shRNA is at leastabout 2-6 fold higher than that of said siRNA.

Another aspect of the invention provides a method of designing a shorthairpin RNA (shRNA) construct for RNAi, said shRNA comprising a 3′overhang of about 1-4 nucleotides, the method comprising selecting thenucleotide about 21 bases 5′ to the most 3′-end nucleotide as the firstpaired nucleotide in a cognate doubled-stranded siRNA with the same 3′overhang.

In one embodiment, the shRNA comprises 25-29 paired polynucleotides.

In one embodiment, the shRNA, when cut by a Dicer enzyme, produces aproduct siRNA that is either identical to, or differ by a singlebasepair immediately 5′ to the 3′ overhang from, said cognate siRNA.

In one embodiment, the Dicer enzyme is a human Dicer.

In one embodiment, the 3′ overhang has 2 nucleotides.

In one embodiment, the shRNA is for RNAi in mammalian cells.

All embodiments described above can be freely combined with one or moreother embodiments whenever appropriate. Such combination also includesembodiments described under different aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RNAi in S2 cells. (a) Drosophila S2 cells were transfected witha plasmid that directs lacZ expression from the copia promoter incombination with dsRNAs corresponding to either human CD8 or lacZ, orwith no dsRNA, as indicated. (b) S2 cells were co-transfected with aplasmid that directs expression of a GFP-US9 fusion protein and dsRNAsof either lacZ or cyclin E, as indicated. Upper panels show FACSprofiles of the bulk population. Lower panels show FACS profiles fromGFP-positive cells. (c) Total RNA was extracted from cells transfectedwith lacZ, cyclin E, fizzy or cyclin A dsRNAs, as indicated. Northernblots were hybridized with sequences not present in the transfecteddsRNAs.

FIG. 2: RNAi in vitro. (a) Transcripts corresponding to either the first600 nucleotides of Drosophila cyclin E (E600) or the first 800nucleotides of lacZ (Z800) were incubated in lysates derived from cellsthat had been transfected with either lacZ or cyclin E (cycE) dsRNAs, asindicated. Time points were 0, 10, 20, 30, 40 and 60 min for cyclin Eand 0, 10, 20, 30 and 60 min for lacZ. (b) Transcripts were incubated inan extract of S2 cells that had been transfected with cyclin E dsRNA(cross-hatched box, below). Transcripts corresponded to the first 800nucleotides of lacZ or the first 600, 300, 220 or 100 nucleotides ofcyclin E, as indicated. Eout is a transcript derived from the portion ofthe cyclin E cDNA not contained within the transfected dsRNA. E-ds isidentical to the dsRNA that had been transfected into S2 cells. Timepoints were 0 and 30 min. (c) Synthetic transcripts complementary to thecomplete cyclin E cDNA (Eas) or the final 600 nucleotides (Eas600) or300 nucleotides (Eas300) were incubated in extract for 0 or 30 min.

FIG. 3: Substrate requirements of the RISC. Extracts were prepared fromcells transfected with cyclin E dsRNA. Aliquots were incubated for 30min at 30° C. before the addition of either the cyclin E (E600) or lacZ(Z800) substrate. Individual 20 μl aliquots, as indicated, werepre-incubated with 1 mM CaCl₂ and 5 mM EGTA, 1 mM CaCl₂, 5 mM EGTA and60 U of micrococcal nuclease, 1 mM CaCl₂ and 60 U of micrococcalnuclease or 10 U of DNase I (Promega) and 5 mM EGTA. After the 30 minpre-incubation, EGTA was added to those samples that lacked it. YeasttRNA (1 μg) was added to all samples. Time points were at 0 and 30 min.

FIG. 4: The RISC contains a potential guide RNA. (a) Northern blots ofRNA from either a crude lysate or the S100 fraction (containing thesoluble nuclease activity, see Methods) were hybridized to a riboprobederived from the sense strand of the cyclin E mRNA. (b) Solublecyclin-E-specific nuclease activity was fractionated as described inMethods. Fractions from the anion-exchange resin were incubated with thelacZ, control substrate (upper panel) or the cyclin E substrate (centrepanel). Lower panel, RNA from each fraction was analysed by northernblotting with a uniformly labeled transcript derived from sense strandof the cyclin E cDNA. DNA oligonucleotides were used as size markers.

FIG. 5: Generation of 22 mers and degradation of mRNA are carried out bydistinct enzymatic complexes. (a) Extracts prepared either from 0-12hour Drosophila embryos or Drosophila S2 cells (see Methods) wereincubated for 0, 15, 30, or 60 minutes (left to right) with auniformly-labeled double-stranded RNA corresponding to the first 500nucleotides of the Drosophila cyclin E coding region. M indicates amarker prepared by in vitro transcription of a synthetic template. Thetemplate was designed to yield a 22 nucleotide transcript. The doubletmost probably results from improper initiation at the +1 position. (b)Whole-cell extracts were prepared from S2 cells that had beentransfected with a dsRNA corresponding to the first 500 nt. of theluciferase coding region. S10 extracts were spun at 30,000×g for 20minutes which represents our standard RISC extract. S100 extracts wereprepared by further centrifugation of S10 extracts for 60 minutes at100,000×g. Assays for mRNA degradation were carried out as describedpreviously for 0, 30 or 60 minutes (left to right in each set) witheither a single-stranded luciferase mRNA or a single-stranded cyclin EmRNA, as indicated. (c) S10 or S100 extracts were incubated with cyclinE dsRNAs for 0, 60 or 120 minutes (L to R).

FIG. 6: Production of 22 mers by recombinant CG4792/Dicer. (a)Drosophila S2 cells were transfected with plasmids that direct theexpression of T7-epitope tagged versions of Drosha, CG4792/Dicer-1 andHomeless. Tagged proteins were purified from cell lysates byimmunoprecipitation and were incubated with cyclin E dsRNA. Forcomparison, reactions were also performed in Drosophila embryo and S2cell extracts. As a negative control, immunoprecipitates were preparedfrom cells transfected with a β-galactosidase expression vector. Pairsof lanes show reactions performed for 0 or 60 minutes. The syntheticmarker (M) is as described in the legend to FIG. 1. (b) Diagrammaticrepresentations of the domain structures of CG4792/Dicer-1, Drosha andHomeless are shown. (c) Immunoprecipitates were prepared from detergentlysates of S2 cells using an antiserum raised against the C-terminal 8amino acids of Drosophila Dicer-1 (CG4792). As controls, similarpreparations were made with a pre-immune serum and with an immune serumthat had been pre-incubated with an excess of antigenic peptide.Cleavage reactions in which each of these precipitates was incubatedwith an ˜500 nt. fragment of Drosophila cyclin E are shown. Forcomparison, an incubation of the substrate in Drosophila embryo extractwas electrophoresed in parallel. (d) Dicer immunoprecipitates wereincubated with dsRNA substrates in the presence or absence of ATP. Forcomparison, the same substrate was incubated with S2 extracts thateither contained added ATP or that were depleted of ATP using glucoseand hexokinase (see methods). (e) Drosophila S2 cells were transfectedwith uniformly, ³²P-labelled dsRNA corresponding to the first 500 nt. ofGFP. RISC complex was affinity purified using a histidine-tagged versionof Drosophila Ago-2, a recently identified component of the RISC complex(Hammond et al., in prep). RISC was isolated either under conditions inwhich it remains ribosome associated (Is, low salt) or under conditionsthat extract it from the ribosome in a soluble form (hs, high salt). Forcomparison, the spectrum of labeled RNAs in the total lysate is shown.(f) Guide RNAs produced by incubation of dsRNA with a Dicerimmunoprecipitate are compared to guide RNAs present in anaffinity-purified RISC complex. These precisely co-migrate on a gel thathas single-nucleotide resolution. The lane labeled control is anaffinity selection for RISC from a cell that had been transfected withlabeled dsRNA but not with the epitope-tagged Drosophila Ago-2.

FIG. 7: Dicer participates in RNAi. (a) Drosophila S2 cells weretransfected with dsRNAs corresponding to the two Drosophila Dicers(CG4792 and CG6493) or with a control dsRNA corresponding to murinecaspase 9. Cytoplasmic extracts of these cells were tested for Diceractivity. Transfection with Dicer dsRNA reduced activity in lysates by7.4-fold. (b) The Dicer-1 antiserum (CG4792) was used to prepareimmunoprecipitates from S2 cells that had been treated as describedabove. Dicer dsRNA reduced the activity of Dicer-1 in this assay by6.2-fold. (c) Cells that had been transfected two days previously witheither mouse caspase 9 dsRNA or with Dicer dsRNA were cotransfected witha GFP expression plasmid and either control, luciferase dsRNA or GFPdsRNA. Three independent experiments were quantified by FACS. Acomparison of the relative percentage of GFP-positive cells is shown forcontrol (GFP plasmid plus luciferase dsRNA) or silenced (GFP plasmidplus GFP dsRNA) populations in cells that had previously beentransfected with either control (caspase 9) or Dicer dsRNAs.

FIG. 8: Dicer is an evolutionarily conserved ribonuclease. (a) A modelfor production of 22 mers by Dicer. Based upon the proposed mechanism ofaction of Ribonuclease III, we propose that Dicer acts on its substrateas a dimer. The positioning of the two ribonuclease domains (RIIIa andRIIIb) within the enzyme would thus determine the size of the cleavageproduct. An equally plausible alternative model could be derived inwhich the RIIIa and RIIIb domains of each Dicer enzyme would cleave inconcert at a single position. In this model, the size of the cleavageproduct would be determined by interaction between two neighboring Dicerenzymes. (b) Comparison of the domain structures of potential Dicerhomologs in various organisms (Drosophila—CG4792, CG6493, C.elegans—K12H4.8, Arabidopsis—CARPEL FACTORY, T25K16.4, AC012328_(—)1,human Helicase-MOI and S. pombe—YC9A_SCHPO). The ZAP domains wereidentified both by analysis of individual sequences with Pfam and byPsi-blast searches. The ZAP domain in the putative S. pombe Dicer is notdetected by PFAM but is identified by Psi-Blast and is thus shown in adifferent color. For comparison, a domain structure of theRDE1/QDE2/ARGONAUTE family is shown. It should be noted that the ZAPdomains are more similar within each of the Dicer and ARGONAUTE familiesthan they are between the two groups. (c) An alignment of the ZAPdomains in selected Dicer and Argonaute family members is shown. Thealignment was produced using ClustalW.

FIG. 9: Purification strategy for RISC. (second step in RNAi model).

FIG. 10: Fractionation of RISC activity over sizing column. Activityfractionates as 500 KDa complex. Also, antibody to Drosophila argonaute2 cofractionates with activity.

FIGS. 11-13: Fractionation of RISC over monoS, monoQ, Hydroxyapatitecolumns. Drosophila argonaute 2 protein also cofactionates.

FIG. 14: Alignment of Drosophila argonaute 2 with other family members.

FIG. 15: Confirmation of Drosophila argonaute 2. S2 cells weretransfected with labeled dsRNA and His tagged argonaute. Argonaute wasisolated on nickel agarose and RNA component was identified on 15%acrylamide gel.

FIG. 16: S2 cell and embryo extracts were assayed for 22-mer generatingactivity.

FIG. 17: RISC can be separated from 22-mer generating activity (dicer).Spinning extracts (S100) can clear RISC activity from supernatant (leftpanel) however, S100 spins still contain dicer activity (right panel).

FIG. 18: Dicer is specific for dsRNA and prefers longer substrates.

FIG. 19: Dicer was fractionated over several columns.

FIG. 20: Identification of dicer as enzyme which can process dsRNA into22 mers. Various RNaseIII family members were expressed with n terminaltags, immunoprecipitated, and assayed for 22-mer generating activity(left panel). In right panel, antibodies to dicer could also precipitate22-mer generating activity.

FIG. 21: Dicer requires ATP.

FIG. 22: Dicer produces RNAs that are the same size as RNAs present inRISC.

FIG. 23: Human dicer homolog when expressed and immunoprecipitated has22-mer generating activity.

FIG. 24: Sequence of Drosophila argonaute 2 (SEQ ID NO: 5). Peptidesidentified by microsequencing are shown in underline.

FIG. 25: Molecular characterization of Drosophila argonaute 2. Thepresence of an intron in coding sequence was determined by northernblotting using intron probe. This results in a different 5′ readingframe then the published genome sequence. Number of polyglutaminerepeats was determined by genomic PCR.

FIG. 26: Dicer activity can be created in human cells by expression ofhuman dicer gene. Host cell was 293. Crude extracts had dicer activity,while activity was absent from untransfected cells. Activity is notdissimilar to that seen in Drosophila embryo extracts.

FIG. 27: A ˜500 nt. fragment of the gene that is to be silenced (X) isinserted into the modified vector as a stable direct repeat usingstandard cloning procedures. Treatment with commercially available crerecombinase reverses sequences within the loxP sites (L) to create aninverted repeat. This can be stably maintained and amplified in an sbcmutant bacterial strain (DL759). Transcription in vitro from thepromoter of choice (P) yields a hairpin RNA that causes silencing. Azeocin resistance marker is included to insure maintenance of the directand inverted repeat structures; however this is non-essential in vitroand could be removed by pre-mRNA splicing if desired. (Smith et al.(2000) Nature 407: 319-20).

FIG. 28: RNAi in P19 embryonal carcinoma cells. Ten-centimeter plates ofP19 cells were transfected by using 5 μg of GFP plasmid and 40 μg of theindicated dsRNA (or no RNA). Cells were photographed by fluorescent(tope panel) and phase-contrast microscopy (bottom panel) at 72 h aftertransfection; silencing was also clearly evident at 48 hpost-transfection.

FIG. 29: RNAi of firefly and Renilla luciferase in P19 cells. (A and B)P19 cells were transfected with plasmids that direct the expression offirefly and Renilla luciferase and dsRNA 500 mers (25 or 250 ng, asindicated in A and B, respectively), that were either homologous to thefirefly luciferase mRNA (dsFF) or nonhomologous (dsGFP). Luciferaseactivities were assayed at various times after transfection, asindicated. Ratios of firefly to Renilla activity are normalized to dsGFPcontrols. (C and D) P19 cells in 12-well culture dishes (2 ml of media)were transfected with 0.25 μg of a 9:1 mix of pGL3-Control and pRL-SV40as well as 2 μg of the indicated RNA. Extracts were prepared 9 h aftertransfection. (C) Ratio of firefly to Renilla luciferase is shown. (D)Ratio of Renilla to firefly luciferase is shown. Values are normalizedto dsGFP. The average of three independent experiments is shown; errorbars indicate standard deviation.

FIG. 30: The panels at the right show expression of either RFP or GFPfollowing transient transfection into wild type P19 cells. The panels atthe left demonstrate the specific suppression of GFP expression in P19clones which stably express a 500 nt double stranded GFP hairpin. P19clones which stably express the double stranded GFP hairpin weretransiently transfected with RFP or GFP, and expression of RFP or GFPwas assessed by visual inspection.

FIG. 31: Specific silencing of luciferase expression by dsRNA in murineembryonic stem cells. Mouse embryonic stem cells in 12-well culturedishes (1 ml of media) were transfected with 1.5 μg of dsRNA along with0.25 μg of a 10:1 mixture of the reporter plasmids pGL3-Control andpRL-SV40. Extracts were prepared and assayed 20 h after transfection.The ratio of firefly to Renilla luciferase expression is shown for FFds500; the ratio of Renilla to firefly is shown for Ren ds500. Both arenormalized to ratios from the dsGFP transfection. The average of threeindependent experiments is shown; error bars indicate standarddeviation.

FIG. 32: RNAi in C2C12 murine myoblast cells. (A) Mouse C2C12 cells in12-well culture dishes (1 ml of media) were transfected with 1 μg of theindicated dsRNA along with 0.250 μg of the reporter plasmidspGL3-Control and pRL-SV40. Extracts were prepared and assayed 24 h aftertransfection. The ratio of firefly to Renilla luciferase expression isshown; values are normalized to ratios from the no dsRNA control. Theaverage of three independent experiments is shown; error bars indicatestandard deviation. (B) C2C12 cells cotransfected with 1 μg of eitherplasmid alone or a plasmid containing a hyperactive mutant of vacciniavirus K3L (Kawagishi-Kobayashi et al. 2000, Virology 276: 424-434). Theabsolute counts of Renilla and firefly luciferase activity are shown.(C) The ratios of firefly/Renilla activity from B, normalized to nodsRNA controls.

FIG. 33: Hela, Chinese hamster ovary, and P19 (pluripotent, mouseembryonic carcinoma) cell lines transfected with plasmids expressingPhotinus pyralis (firefly) and Renilla reniformis (sea pansy)luciferases and with dsRNA 500 mers (400 ng), homologous to eitherfirefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Dualluciferase assays were carried out using an Analytical ScientificInstruments model 3010 Luminometer. In this assay Renilla luciferaseserves as an internal control for dsRNA-specific suppression of fireflyluciferase activity. These data demonstrate that 500-mer dsRNA canspecifically suppress cognate gene expression in vitro.

FIG. 34: Expression of a hairpin RNA produces P19 EC cell lines thatstably silence GFP. (A) A cartoon of the FLIP cassette used to constructthe GFP hairpin. GFP represents the first 500 coding base pairs of EGFP.Zeo, zeocin resistance gene; L, Lox; P, the cytomegalovirus promoter inthe expression plasmid pcDNA3. Homologous GFP fragments are first clonedas direct repeats into the FLIP cassette. To create inverted repeats forhairpin production, the second repeat is flipped by using Crerecombinase. When transcribed, the inverted repeat forms a GFP dsRNAwith a hairpin loop. (B) P19 cell lines stably expressing the GFPhairpin plasmid, GFPhp.1 (clone 10) and GFPhp.2 (clone 12), along withwt P19 were transfected with 0.25 μg each of GFP and RFP reporter genes.Fluorescence micrographs were taken by using filters appropriate for GFPand RFP. Magnification is 200×. (C) P19 GFPhp.1 cells were transfectedwith pEGFP and 0, 0.5, or 1 μg of Dicer or firefly dsRNA. Fluorescencemicrographs were taken at 48 h post-transfection and are superimposedwith bright field images to reveal non-GFP expressing cells.Magnification is 100×. (D) In vitro and in vitro processing of dsRNA inP19 cells. In vitro Dicer assays were performed on S2 cells and threeindependently prepared P19 extracts by using ³²P-labeled dsRNA (30° C.for 30 min). A Northern blot of RNA extracted from control and GFPhp.1P19 cells shows the production of ≈22-mer RNA species inhairpin-expressing cells but not in control cells. Blots were probedwith a ³²P-labeled “sense” GFP transcript.

FIG. 35: dsRNA induces silencing at the posttranscriptional level. P19cell extracts were used for in vitro translation of firefly and Renillaluciferase mRNA (100 ng each). Translation reactions were programmedwith various amounts of dsRNA 500 mers, either homologous to fireflyluciferase mRNA (dsLUC) or nonhomologous (dsGFP). Luciferase assays werecarried out after a 1 h incubation at 30° C. Ratios of firefly toRenilla activity are normalized to no dsRNA controls. Standarddeviations from the mean are shown.

FIG. 36: S10 fractions from P19 cell lysates were used for in vitrotranslations of mRNA coding for Photinus pyralis (firefly) and Renillareniformis (sea pansy) luciferases. Translation reactions wereprogrammed with dsRNA, ssRNA, or asRNA 500 mers, either complementary tofirefly luciferase mRNA (dsFF, ssFF, or asFF), complementary to Renillaluciferase (dsREN, ssREN, or asREN) or non-complementary (dsGFP).Reactions were carried out at 30° C. for 1 hour, after a 30 minpreincubation with dsRNA, ssRNA, or asRNA. Dual luciferase assays werecarried out using an Analytical Scientific Instruments model 3010Luminometer. On the left, Renilla luciferase serves as an internalcontrol for dsRNA-specific suppression of firefly luciferase activity.On the right, firefly luciferase serves as an internal control fordsRNA-specific suppression of Renilla luciferase activity. These datademonstrate that 500-mer double-stranded RNA (dsRNA) but notsingle-stranded (ssRNA) or anti-sense RNA (asRNA) suppresses cognategene expression in vitro in a manner consistent withpost-transcriptional gene silencing.

FIG. 37: P19 cells were grown in 6-well tissue culture plates toapproximately 60% confluence. Various amounts of dsRNA, eitherhomologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP),were added to each well and incubated for 12 hrs under normal tissueculture conditions. Cells were then transfected with plasmids expressingPhotinus pyralis (firefly) and Renilla reniformis (sea pansy)luciferases and with dsRNA 500 mers (500 ng). Dual luciferase assayswere carried out 12 hrs post-transfection using an Analytical ScientificInstruments model 3010 Luminometer. In this assay Renilla luciferaseserves as an internal control for dsRNA-specific suppression of fireflyluciferase activity. These data show that 500-mer dsRNA can specificallysuppress cognate gene expression in vitro without transfection undernormal tissue culture conditions.

FIG. 38: Previous methods for generating siRNAs required costly chemicalsynthesis. The invention provides an in vitro method for synthesizingsiRNAs using standard RNA transcription reactions.

FIG. 39: Short hairpins suppress gene expression in Drosophila S2 cells.(A) Sequences and predicted secondary structure of representativechemically synthesized RNAs. Sequences correspond to positions 112-134(siRNA) and 463-491 (shRNAs) of Firefly luciferase carried onpGL3-Control. An siRNA targeted to position 463-485 of the luciferasesequence was virtually identical to the 112-134 siRNA in suppressingexpression, but is not shown. These sequences are represented by SEQ IDNOs: 6-10. (B) Exogenously supplied short hairpins suppress expressionof the targeted Firefly luciferase gene in vitro. Six-well plates of S2cells were transfected with 250 ng/well of plasmids that direct theexpression of firefly and Renilla luciferase and 500 ng/well of theindicated RNA. Luciferase activities were assayed 48 h aftertransfection. Ratios of firefly to Renilla luciferase activity werenormalized to a control transfected with an siRNA directed at the greenfluorescent protein (GFP). The average of three independent experimentsis shown; error bars indicate standard deviation. (C) Short hairpins areprocessed by the Drosophila Dicer enzyme. T7 transcribed hairpinsshFfL22, shFfL29, and shFfS29 were incubated with (+) and without (−)0-2-h Drosophila embryo extracts. Those incubated with extract produced˜22-nt siRNAs, consistent with the ability of these hairpins to induceRNA interference. A long dsRNA input (cyclin E 500-mer) was used as acontrol. Cleavage reactions were performed as described in Bernstein etal., 2001, Nature, 409:363-366.

FIG. 40: Short hairpins function in mammalian cells. HEK 293T, HeLa,COS-1, and NIH 3T3 cells were transfected with plasmids and RNAs as inFIG. 1 and subjected to dual luciferase assays 48 h post-transfection.The ratios of firefly to Renilla luciferase activity are normalized to acontrol transfected with an siRNA directed at the green fluorescentprotein (GFP). The average of three independent experiments is shown;error bars indicate standard deviation.

FIG. 41: siRNAs and short hairpins transcribed in vitro suppress geneexpression in mammalian cells. (A) Sequences and predicted secondarystructure of representative in vitro transcribed siRNAs. Sequencescorrespond to positions 112-134 (siRNA) and 463-491 (shRNAs) of fireflyluciferase carried on pGL3-Control. These sequences are represented bySEQ ID NOs: 11-20. (B) In vitro transcribed siRNAs suppress expressionof the targeted firefly luciferase gene in vitro. HEK 293T cells weretransfected with plasmids as in FIG. 2. The presence of non-base-pairedguanosine residues at the 5′ end of siRNAs significantly alters thepredicted end structure and abolishes siRNA activity. (C) Sequences andpredicted secondary structure of representative in vitro transcribedshRNAs. Sequences correspond to positions 112-141 of firefly luciferasecarried on pGL3-Control. These sequences are represented by SEQ ID NOs:21-26. (D) Short hairpins transcribed in vitro suppress expression ofthe targeted firefly luciferase gene in vitro. HEK 293T cells weretransfected with plasmids as in FIG. 2.

FIG. 42: Transcription of functional shRNAs in vitro. (A) Schematic ofthe pShh1 vector. Sequences encoding shRNAs with between 19 and 29 basesof homology to the targeted gene are synthesized as 60-75-bpdouble-stranded DNA oligonucleotides and ligated into an EcoRV siteimmediately downstream of the U6 promoter. This sequence is representedby SEQ ID NO: 27. (B) Sequence and predicted secondary structure of theFf1 hairpin. (C) An shRNA expressed from the pShh1 vector suppressesluciferase expression in mammalian cells. HEK 293T, HeLa, COS-1, and NIH3T3 cells were transfected with reporter plasmids as in FIG. 1, andpShh1 vector, firefly siRNA, or pShh1 firefly shRNA constructs asindicated. The ratios of firefly to Renilla luciferase activity weredetermined 48 h after transfection and represent the average of threeindependent experiments; error bars indicate standard deviation.

FIG. 43: Dicer is required for shRNA-mediated gene silencing. HEK 293Tcells were transfected with luciferase reporter plasmids as well aspShh1-Ff1 and an siRNA targeting human Dicer either alone or incombination, as indicated. The Dicer siRNA sequence(TCAACCAGCCACTGCTGGA, SEQ ID NO: 37) corresponds to coordinates3137-3155 of the human Dicer sequence. The ratios of firefly to Renillaluciferase activity were determined 26 h after transfection andrepresent the average of three independent experiments; error barsindicate standard deviation.

FIG. 44: Stable shRNA-mediated gene silencing of an endogenous gene. (A)Sequence and predicted secondary structure of the p53 hairpin. The 5′shRNA stem contains a 27-nt sequence derived from mouse p53 (nucleotides166-192), whereas the 3′ stem harbors the complimentary antisensesequence. This sequence is represented by SEQ ID NO: 28. (B) Senescencebypass in primary mouse embryo fibroblasts (MEFs) expressing an shRNAtargeted at p53. Wild-type MEFs, passage 5, were transfected withpBabe-RasV12 with control plasmid or with p53hp (5 μg each with FuGENE;Roche). Two days after transfection, cells were trypsinized, counted,and plated at a density of 1×10⁵/10-cm plate in media containing 2.0μg/mL of puromycin. Control cells cease proliferation and show asenescent morphology (left panel). Cells expressing the p53 hairpincontinue to grow (right panel). Photos were taken 14 dpost-transfection.

FIG. 45: A mixture of two short hairpins, both corresponding to fireflyluciferase, does not result in a synergistic suppression of geneexpression. Suppression of firefly luciferase gene expression resultingfrom transfection of a mixture of two different short hairpins (HP #1and HP #2) was examined. The mixture of HP #1 and HP #2 did not have amore robust effect on the suppression of firefly luciferase geneexpression than expression of HP #1 alone.

FIG. 46: Encoded short hairpins specifically suppress gene expression invitro. DNA oligonucleotides encoding 29 nucleotide hairpinscorresponding to firefly luciferase were inserted into a vectorcontaining the U6 promoter. Three independent constructs were examinedfor their ability to specifically suppress firefly luciferase geneexpression in 293T cells. siOligo1-2, siOligo1-6, and siOligo1-19(construct in the correct orientation) each suppressed gene expressionas effectively as siRNA. In contrast, siOligo1-10 (construct in theincorrect orientation) did not suppress gene expression. An independentconstruct targeted to a different portion of the firefly luciferase genedid not effectively suppress gene expression in either orientation(siOligo2-23, siOligo2-36).

FIGS. 47-49: Strategies for stable expression of short dsRNAs.

FIG. 50: Dual luciferase assays were performed as described in detail inFIGS. 28-35, however the cells used in these experiments were PKR^(−/−)murine embryonic fibroblasts (MEFs). Briefly, RNAi using long dsRNAstypically envokes a non-specific response in MEFs (due to PKR activity).To evaluate the effect of long dsRNA constructs to specifically inhibitgene expression in MEFs, RNAi was examined in PKR^(−/−) MEFs. Such cellsdo not respond to dsRNA with a non-specific response. The datasummarized in this figure demonstrates that in the absence of thenon-specific PKR response, long dsRNA constructs specifically suppressgene expression in MEFs.

FIG. 51: Is a schematic representation of the mouse tyrosinase promoter.Primers were used to amplify three separate regions in the proximalpromoter, or to amplify sequence corresponding to an enhancer locatedapproximately 12 kb upstream.

FIG. 52: Reporter expression plasmids and siRNA sequences used inFigures X and Y. PGL-3-Control and Pluc-NS5B are the expression plasmidsused for transfection into mouse liver. The nucleotide sequences of thesiRNAs used in the study are shown underneath. These sequences arerepresented by SEQ ID NOs: 29-35.

FIG. 53: RNA interference in adult mice using siRNAs. (a) Representativeimages of light emitted from mice co-transfected with the luciferaseplasmid pGL3-control and either no siRNA, luciferase siRNA or unrelatedsiRNA. A pseudocolour image representing intensity of emitted light(red, most intense; blue, least intense) superimposed on a greyscalereference image (for orientation) shows that RNAi functions in adultmice. Annealed 21-nucleotide siRNAs (40 μg; Dharmacon) were co-injectedinto the livers of mice with 2 μg pGL3-control DNA (Promega) and 800units of RNasin (Promega) in 1.8 ml PBS buffer in 5-7 s. After 72 h,mice were anaesthetized and given 3 mg luciferin intraperitoneally 15min before imaging. (b) siRNA results (six mice per group) from arepresentative experiment. Mice receiving luciferase siRNA emittedsignificantly less light than reporter-alone controls (one-way ANOVAwith post hoc Fisher's test). Results for reporter alone and unrelatedsiRNA were statistically similar. Animals were treated according to theUS National Institutes of Health's guidelines for animal care and theguidelines of Stanford University.

FIG. 54: RNA interference in adult mice using shRNAs. (a) Representativeimages of light emitted from mice co-transfected with the luciferaseplasmid control, pShh1-Ff1, and pShh1-Ff1rev. pShh1-Ff1, but notpShh1-Ff1rev, reduced luciferase expression in mice relative to thereporter-alone control. pShh1-Ff1 or pShh1-rev (10 μg) were co-injectedwith 2 μg pGL3-control in 1.8 ml PBS buffer. (b) Average of threeindependent shRNA experiments (n=5). Average values for thereporter-alone group are designated as 100% in each of the threeexperiments. Animals were treated according to the US NationalInstitutes of Health's guidelines for animal care and the guidelines ofStanford University.

FIG. 55: Heritable repression of Neil1 expression by RNAi in severaltissues. (a) Expression of Neil1 mRNA in the livers of three micecontaining the Neil1 shRNA transgene (shRNA-positive) or three siblingslacking the transgene (shRNA-negative) was assayed by RT-PCR (top row isNeil1). An RT-PCR of β-actin was done to ensure that equal quantities ofmRNAs were tested for each mouse (second row). Expression of theneomycin resistance gene (neo), carried on the shRNA vector, was testedsimilarly (third row). Finally, the mice were genotyped using genomicDNA that was PCR-amplified with vector-specific primers (bottom row).(b) Similar studies were performed in the heart. (c) Similar studieswere performed in the spleen. Animal procedures have been approved bythe SUNY, Stony Brook Institutional Animal Care and Use Committee(IACUC).

FIG. 56: Reduction in Neil1 protein correlates with the presence ofsiRNAs. (a) Expression of Neil1 protein was examined in protein extractsfrom the livers of mice carrying the shRNA transgene (shRNA-positive) orsiblings lacking the transgene (shRNA-negative) by western blotting withNeil1-specific antiserum. A western blot for PCNA was used tostandardize loading. (b) The presence of siRNAs in RNA derived from thelivers of transgenic mice as assayed by northern blotting using a 300 ntprobe, part of which was complementary to the shRNA sequence. We notesiRNAs only in mice transgenic for the shRNA expression cassette.

FIG. 57: In vitro processing of 29 nt. shRNAs by Dicer generates asingle siRNA from the end of each short hairpin. a) The set of shRNAscontaining 19 or 29 nt stems and either bearing or lacking a 2nucleotide 3′overhang is depicted schematically. For reference the 29 ntsequence from luciferase (top, blue) strand is given. The presumedcleavage sites are indicated in green and by the arrows. b) In vitroDicer processing of shRNAs. Substrates as depicted in a) were incubatedeither in the presence or absence of recombinant human Dicer (asindicated). Processing of a 500 bp. blunt-ended dsRNA is shown forcomparison. Markers are end-labeled, single-stranded, synthetic RNAoligonucleotides. c) All shRNA substrates were incubated with bacterialRNase III to verify their double-stranded nature. This sequence isrepresented by SEQ ID NO: 36.

FIG. 58: Primer extension analysis reveal a single siRNA generated fromDicer processing of shRNA both in vitro and in vivo. a) 19 nt. shRNAs,as indicated (see FIG. 57 a), were processed by Dicer in vitro. ReactedRNAs were extended with a specific primer that yields a 20 base productif cleavage occurs 22 bases from the 3′ end of the overhung RNA (seeFIG. 57 a). Lanes labeled siRNA are extensions of synthetic RNAscorresponding to predicted siRNAs that would be released by cleavage 21or 22 nucleotides from the 3′ end of the overhung precursor. Observationof extension products dependents entirely on the inclusion of RT(indicated). Markers are phosphorylated, synthetic DNA oligonucleotides.b) Analysis as described in a) for 29 nt. shRNAs. The * indicates thespecific extension product from the overhung shRNA species. c) Primerextension were used to analyze products from processing of overhung 29nt. shRNAs in vivo. For comparison, extensions of in vitro processedmaterial are also shown. Again, the * indicates the specific extensionproduct.

FIG. 59: Gene suppression by shRNAs is comparable to or more effectivethan that achieved by siRNAs targeting the same sequences. a) Structuresof synthetic RNAs used for these studies. b) mRNA suppression levelsachieved by 43 siRNAs targeting 6 different genes compared with levelsachieved by 19-mer (left) or 29-mer (right) shRNAs derived from the sametarget sequences. All RNAs were transfected at a final concentration of100 nM. Values indicated on the X and Y axes reflect the percentage ofmRNA remaining in HeLa cells 24 hours after RNA transfection comparedwith cells treated with transfection reagent alone. c) Titrationanalysis comparing efficacies of four siRNA/shRNA sets targeting MAPK14.Curves are graphed from data derived from transfections at 1.56, 6.25,25, and 100 nM final concentrations of RNA. (diamonds: 21-mer siRNAs;squares: 19-mer shRNAs; triangles: 29-mer shRNAs).

FIG. 60: Microarray profiling reveals sequence-specific gene expressionprofiles and more similarity between 29-mer shRNAs and cognate siRNAsthan observed for 19-mer shRNAs. Each row of the heat maps reports thegene expression signature resulting from transfection of an individualRNA. Data shown represent genes that display at least a 2-fold change inexpression level (P value<0.01 and log10 intensity>1) relative tomock-transfected cells. Green indicates decreased expression relative tomock transfection whereas red indicates elevated expression. a) 19-mershRNAs and siRNAs designed for six different target sequences within thecoding region of the MAPK14 gene were tested for gene silencing after 24hours in HeLa cells. b) A similar experiment to that described in a) butcarried out with five 29-mer shRNAs targeting MAPK14.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS I. OVERVIEW

The present invention provides methods for attenuating gene expressionin a cell using gene-targeted double stranded RNA (dsRNA). The dsRNAcontains a nucleotide sequence that hybridizes under physiologicconditions of the cell to the nucleotide sequence of at least a portionof the gene to be inhibited (the “target” gene). The nucleotide sequencecan hybridize to either coding or non-coding sequence of the targetgene.

A significant aspect to certain embodiments of the present inventionrelates to the demonstration in the present application that RNAi can infact be accomplished both in cultured mammalian cells and in wholeorganisms. This had not been previously described in the art.

Another salient feature of the present invention concerns the ability tocarry out RNAi in higher eukaryotes, particularly in non-oocytic cellsof mammals, e.g., cells from adult mammals as an example.

Furthermore, in contrast to the teachings of the prior art, wedemonstrate that RNAi in mammalian systems can be mediated with dsRNAidentical or similar to non-coding sequence of a target gene. It waspreviously believed that although dsRNA identical or similar tonon-coding sequences (i.e., promoter, enhancer, or intronic sequences)did not inhibit RNAi, such dsRNAs were not thought to mediate RNAi.

In addition, the instant invention also demonstrates that short hairpinRNA (shRNA) may effectively be used in the subject RNAi methods. Incertain embodiments, shRNAs specifically designed as Dicer substratescan be used as more potent inducers of RNAi than siRNAs. Not only ismaximal inhibition achieved at much lower levels of transfected RNA, butalso endpoint inhibition is often greater. In certain other embodiments,mimicking natural pre-miRNAs by inclusion of a 1-5 nucleotide(s),especially a 2 nucleotide 3′ overhang, enhances the efficiency of Dicercleavage and directs cleavage to a specific position in the precursor.The presence of this specific processing site further permits theapplication of rules for siRNA design to shRNAs, both for chemicalsynthesis and vector-based delivery of such shRNA constructs. Theseteachings provide improved methods for evoking RNAi in mammalian cells,and thus improved ability to produce highly potent silencing triggers intherapeutic application of RNAi.

As described in further detail below, the present invention(s) are basedon the discovery that the RNAi phenomenon is mediated by a set of enzymeactivities, including an essential RNA component, that areevolutionarily conserved in eukaryotes ranging from plants to mammals.

One enzyme contains an essential RNA component. After partialpurification, a multi-component nuclease (herein “RISC nuclease”)co-fractionates with a discrete, 22-nucleotide RNA species which mayconfer specificity to the nuclease through homology to the substratemRNAs. The short RNA molecules are generated by a processing reactionfrom the longer input dsRNA. Without wishing to be bound by anyparticular theory, these 22-mer guide RNAs may serve as guide sequencesthat instruct the RISC nuclease to destroy specific mRNAs correspondingto the dsRNA sequences.

As illustrated, double stranded forms of the 22-mer guide RNA can besufficient in length to induce sequence-dependent dsRNA inhibition ofgene expression. In the illustrated example, dsRNA constructs areadministered to cells having a recombinant luciferase reporter gene. Inthe control cell, e.g., no exogeneously added RNA, the level ofexpression of the luciferase reporter is normalized to be the value of“1”. As illustrated, both long (500-mer) and short (22-mer) dsRNAconstructs complementary to the luciferase gene could inhibit expressionof that gene product relative to the control cell. On the other hand,similarly sized dsRNA complementary to the coding sequence for anotherprotein, green fluorescence protein (GFP), did not significantly effectthe expression of luciferase—indicating that the inhibitory phenomenawas in each case sequence-dependent. Likewise, single stranded 22-mersof luciferase did not inhibit expression of that gene—indicating thatthe inhibitory phenomena is double stranded-dependent.

The appended examples also identify an enzyme, Dicer, that can producethe putative guide RNAs. Dicer is a member of the RNAse III family ofnucleases that specifically cleave dsRNA and is evolutionarily conservedin worms, flies, plants, fungi and, as described herein, mammals. Theenzyme has a distinctive structure which includes a helicase domain anddual RNAse III motifs. Dicer also contains a region of homology to theRDE1/QDE2/ARGONAUTE family, which have been genetically linked to RNAiin lower eukaryotes. Indeed, activation of, or overexpression of Dicermay be sufficient in many cases to permit RNA interference in otherwisenon-receptive cells, such as cultured eukaryotic cells, or mammalian(non-oocytic) cells in culture or in whole organisms.

In certain embodiments, the cells can be treated with an agent(s) thatinhibits the general double-stranded RNA response(s) by the host cells,such as may give rise to sequence-independent apoptosis. For instance,the cells can be treated with agents that inhibit the dsRNA-dependentprotein kinase known as PKR (protein kinase RNA-activated). Doublestranded RNAs in mammalian cells typically activate protein kinase PKRand lead to apoptosis. The mechanism of action of PKR includesphosphorylation and inactivation of eIF2α (Fire, Trends Genet 15: 358,1999). It has also been reported that induction of NF-κB by PKR isinvolved in apoptosis commitment and this process is mediated throughactivation of the IKK complex. This sequence-independent response mayreflect a form of primitive immune response, since the presence of dsRNAis a common feature of many viral lifecycles.

As described herein, Applicants have demonstrated that the PKR responsecan be overcome in favor of the sequence-specific RNAi response.However, in certain instances, it may be desirable to treat the cellswith agents which inhibit expression of PKR, cause its destruction,and/or inhibit the kinase activity of PKR, and such methods arespecifically contemplated for use in the present invention. Likewise,overexpression of agents which ectopically activate eIF2α can be used.Other agents which can be used to suppress the PKR response includeinhibitors of IKK phosphorylation of IκB, inhibitors of IκBubiquitination, inhibitors of IκB degradation, inhibitors of NF-κBnuclear translocation, and inhibitors of NF-κB interaction with κBresponse elements.

Other inhibitors of sequence-independent dsRNA response in cells includethe gene product of the vaccinia virus E3L. The E3L gene productcontains two distinct domains. A conserved carboxy-terminal domain hasbeen shown to bind double-stranded RNA (dsRNA) and inhibit the antiviraldsRNA response by cells. Expression of at least that portion of the E3Lgene in the host cell, or the use of polypeptide or peptidomimeticsthereof, can be used to suppress the general dsRNA response. Caspaseinhibitors sensitize cells to killing by double-stranded RNA.Accordingly, ectopic expression or activation of caspases in the hostcell can be used to suppress the general dsRNA response.

In other embodiments, the subject method is carried out in cells whichhave little or no general response to double stranded RNA, e.g., have noPKR-dependent dsRNA response, at least under the culture conditions. Asillustrated in FIGS. 28-32, CHO and P19 cells can be used without havingto inhibit PKR or other general dsRNA responses.

Also as described in further detail below, the present invention(s) arepartially based on the discovery that short hairpin RNA specificallydesigned as Dicer substrates are more potent inducers of RNAi thansiRNAs. In certain embodiments, shRNA constructs with 1-5, preferablytwo 3′ overhang nucleotides are substrates particulary well-adpated forDicer-mediated cleavage, and are more potent inhibitors of target genesthen their siRNA counterparts with identical complementary sequences.Such shRNA can be formed either in vitro or in vivo by, for example,sequence-specific pairing after chemical synthesis, or transcriptionfrom a promoter operatively-linked to a DNA encoding such hairpinstructure.

Thus, the present invention provides a process and compositions forinhibiting expression of a target gene in a cell, especially a mammaliancell. In certain embodiments, the process comprises introduction of RNA(the “dsRNA construct”) with partial or fully double-stranded characterinto the cell or into the extracellular environment. Inhibition isspecific in that a nucleotide sequence from a portion of the target geneis chosen to produce the dsRNA construct. The dsRNA may be identical orsimilar to coding or non-coding sequence of the target gene. Inpreferred embodiments, the method utilizes a cell in which Dicer and/orArgonaute activities are recombinantly expressed or otherwiseectopically activated. This process can be (1) effective in attenuatinggene expression, (2) specific to the targeted gene, and (3) general inallowing inhibition of many different types of target gene.

II. DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromosomalDNA of the host cell. Another type of vector is an episomal vector,i.e., a nucleic acid capable of extra-chromosomal replication. Vectorscapable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Inthe present specification, “plasmid” and “vector” are usedinterchangeably unless otherwise clear from the context.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as applicable tothe embodiment being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide ofthe present invention, including both exon and (optionally) intronsequences. The nucleic acid may also optionally include non-codingsequences such as promoter or enhancer sequences. A “recombinant gene”refers to nucleic acid encoding such regulatory polypeptides, that mayoptionally include intron sequences that are derived from chromosomalDNA. The term “intron” refers to a DNA sequence present in a given genethat is not translated into protein and is generally found betweenexons.

A “protein coding sequence” or a sequence that “encodes” a particularpolypeptide or peptide, is a nucleic acid sequence that is transcribed(in the case of DNA) and is translated (in the case of mRNA) into apolypeptide in vitro or in vitro when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

Likewise, “encodes”, unless evident from its context, will be meant toinclude DNA sequences that encode a polypeptide, as the term istypically used, as well as DNA sequences that are transcribed intoinhibitory antisense molecules.

The term “loss-of-function”, as it refers to genes inhibited by thesubject RNAi method, refers to a diminishment in the level of expressionof a gene(s) in the presence of one or more dsRNA construct(s) whencompared to the level in the absence of such dsRNA construct(s).

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein coding sequence results fromtranscription and translation of the coding sequence.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “cultured cells” refers to cells suspended in culture, e.g.,dispersed in culture or in the form tissue. It does not, however,include oocytes or whole embryos (including blastocysts and the like)which may be provided in culture. In certain embodiments, the culturedcells are adults cells, e.g., non-embryonic.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

As used herein, the terms “transduction” and “transfection” are artrecognized and mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses adsRNA construct.

“Transient transfection” refers to cases where exogenous DNA does notintegrate into the genome of a transfected cell, e.g., where episomalDNA is transcribed into mRNA and translated into protein.

A cell has been “stably transfected” with a nucleic acid construct whenthe nucleic s acid construct is capable of being inherited by daughtercells.

As used herein, a “reporter gene construct” is a nucleic acid thatincludes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct that isheterologously expressed in a cell.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control. For purposes of this invention, the terms“transformed phenotype of malignant mammalian cells” and “transformedphenotype” are intended to encompass, but not be limited to, any of thefollowing phenotypic traits associated with cellular transformation ofmammalian cells: immortalization, morphological or growthtransformation, and tumorigenicity, as detected by prolonged growth incell culture, growth in semi-solid media, or tumorigenic growth inimmuno-incompetent or syngeneic animals.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical, genetic, and/or recombinant means such that thecells have the ability to grow through an indefinite number of divisionsin culture.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

“MHC antigen”, as used herein, refers to a protein product of one ormore MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes.

The term “histocompatibility” refers to the similarity of tissue betweendifferent individuals. The level of histocompatibility describes howwell matched the patient and donor are. The major histocompatibilitydeterminants are the human leukocyte antigens (HLA). HLA typing isperformed between the potential marrow donor and the potentialtransplant recipient to determine how close a HLA match the two are. Thecloser the match the less the donated marrow and the patient's body willreact against each other.

The term “human leukocyte antigens” or “HLA”, refers to proteins(antigens) found on the surface of white blood cells and other tissuesthat are used to match donor and patient. For instances, a patient andpotential donor may have their white blood cells tested for such HLAantigens as, HLA-A, B and DR. Each individual has two sets of theseantigens, one set inherited from each parent. For this reason, it ismuch more likely for a brother or sister to match the patient than anunrelated individual, and much more likely for persons of the sameracial and ethnic backgrounds to match each other.

III. EXEMPLARY EMBODIMENTS OF ISOLATION METHOD

One aspect of the invention provides a method for potentiating RNAi byinduction or ectopic activation of an RNAi enzyme in a cell (in vitro orin vitro) or cell-free mixtures. In preferred embodiments, the RNAiactivity is activated or added to a mammalian cell, e.g., a human cell,which cell may be provided in vitro or as part of a whole organism. Inother embodiments, the subject method is carried out using eukaryoticcells generally (except for oocytes) in culture. For instance, the Dicerenzyme may be activated by virtue of being recombinantly expressed or itmay be activated by use of an agent which (i) induces expression of theendogenous gene, (ii) stabilizes the protein from degradation, and/or(iii) allosterically modifies the enzyme to increase its activity (byaltering its k_(cat), K_(m) or both).

A. Dicer and Argonaut Activities

In certain embodiments, at least one of the activated RNAi enzymes isDicer, or a homolog thereof. In certain preferred embodiments, thepresent method provides for ectopic activation of Dicer. As used herein,the term “Dicer” refers to a protein which (a) mediates an RNAi responseand (b) has an amino acid sequence at least 50 percent identical, andmore preferably at least 75, 85, 90 or 95 percent identical to SEQ IDNO: 2 or 4, and/or which can be encoded by a nucleic acid whichhybridizes under wash conditions of 2×SSC at 22° C., and more preferably0.2×SSC at 65° C., to a nucleotide represented by SEQ ID NO: 1 or 3.Accordingly, the method may comprise introducing a dsRNA construct intoa cell in which Dicer has been recombinantly expressed or otherwiseectopically activated.

In certain embodiment, at least one of the activated RNAi enzymes isArgonaut, or a homolog thereof. In certain preferred embodiments, thepresent method provides for ectopic activation of Argonaut. As usedherein, the term “Argonaut” refers to a protein which (a) mediates anRNAi response and (b) has an amino acid sequence at least 50 percentidentical, and more preferably at least 75, 85, 90 or 95 percentidentical to the amino acid sequence shown in FIG. 24. Accordingly, themethod may comprise introducing a dsRNA construct into a cell in whichArgonaut has been recombinantly expressed or otherwise ectopicallyactivated.

This invention also provides expression vectors containing a nucleicacid encoding a Dicer or Argonaut polypeptide, operably linked to atleast one transcriptional regulatory sequence. Operably linked isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the subject Dicer or Argonaut proteins. Accordingly, theterm transcriptional regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences aredescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif., 1990. For instance, any of awide variety of expression control sequences, sequences that control theexpression of a DNA sequence when operatively linked to it, may be usedin these vectors to express DNA sequences encoding Dicer or Argonautpolypeptides of this invention. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed.

Moreover, the vector's copy number, the ability to control that copynumber and the expression of any other proteins encoded by the vector,such as antibiotic markers, should also be considered.

The recombinant Dicer or Argonaut genes can be produced by ligating anucleic acid encoding a Dicer or Argonaut polypeptide into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject Dicer or Argonaut polypeptides include plasmids and othervectors. For instance, suitable vectors for the expression of a Dicer orArgonaut polypeptide include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as Ampicillin can beused. In an illustrative embodiment, a Dicer or Argonaut polypeptide isproduced recombinantly utilizing an expression vector generated bysub-cloning the coding sequence of a Dicer or Argonaut gene.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In yet another embodiment, the subject invention provides a “geneactivation” construct which, by homologous recombination with a genomicDNA, alters the transcriptional regulatory sequences of an endogenousDicer or Argonaut gene. For instance, the gene activation construct canreplace the endogenous promoter of a Dicer or Argonaut gene with aheterologous promoter, e.g., one which causes constitutive expression ofthe Dicer or Argonaut gene or which causes inducible expression of thegene under conditions different from the normal expression pattern ofDicer or Argonaut. A variety of different formats for the geneactivation constructs are available. See, for example, the TranskaryoticTherapies, Inc PCT publications WO93/09222, WO95/31560, WO96/29411,WO95/31560 and WO94/12650.

In preferred embodiments, the nucleotide sequence used as the geneactivation construct can be comprised of (1) DNA from some portion ofthe endogenous Dicer or Argonaut gene (exon sequence, intron sequence,promoter sequences, etc.) which direct recombination and (2)heterologous transcriptional regulatory sequence(s) which is to beoperably linked to the coding sequence for the genomic Dicer or Argonautgene upon recombination of the gene activation construct. For use ingenerating cultures of Dicer or Argonaut producing cells, the constructmay further include a reporter gene to detect the presence of theknockout construct in the cell.

The gene activation construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to provide theheterologous regulatory sequences in operative association with thenative Dicer or Argonaut gene. Such insertion occurs by homologousrecombination, i.e., recombination regions of the activation constructthat are homologous to the endogenous Dicer or Argonaut gene sequencehybridize to the genomic DNA and recombine with the genomic sequences sothat the construct is incorporated into the corresponding position ofthe genomic DNA.

The terms “recombination region” or “targeting sequence” refer to asegment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

As used herein, the term “replacement region” refers to a portion of aactivation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

The heterologous regulatory sequences, e.g., which are provided in thereplacement region, can include one or more of a variety of elements,including: promoters (such as constitutive or inducible promoters),enhancers, negative regulatory elements, locus control regions,transcription factor binding sites, or combinations thereof.

Promoters/enhancers which may be used to control the expression of thetargeted gene in vitro include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., J. Exp. Med169: 13, 1989), the human β-actin promoter (Gunning et al., PNAS 84:4831-4835, 1987), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal., Mol. Cell Biol. 4: 1354-1362, 1984), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.,Nature 290: 304-310, 1981; Templeton et al., Mol. Cell Biol. 4: 817,1984; and Sprague et al., J. Virol. 45: 773, 1983), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., Cell 22: 787-797, 1980), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al., PNAS 82:3567-71, 1981), and the herpes simplex virus LAT promoter (Wolfe et al.,Nature Genetics 1: 379-384, 1992).

In still other embodiments, the replacement region merely deletes anegative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

B. Cell/Organism

The cell with the target gene may be derived from or contained in anyorganism (e.g., plant, animal, protozoan, virus, bacterium, or fungus).The dsRNA construct may be synthesized either in vitro or in vitro.Endogenous RNA polymerase of the cell may mediate transcription invitro, or cloned RNA polymerase can be used for transcription in vitroor in vitro. For generating double stranded transcripts from a transgenein vitro, a regulatory region may be used to transcribe the RNA strand(or strands). Furthermore, dsRNA can be generated by transcribing an RNAstrand which forms a hairpin, thus producing a dsRNA.

Genetic manipulation becomes possible in organisms that are notclassical genetic models. Breeding and screening programs may beaccelerated by the ability to rapidly assay the consequences of aspecific, targeted gene disruption. Gene disruptions may be used todiscover the function of the target gene, to produce disease models inwhich the target gene are involved in causing or preventing apathological condition, and to produce organisms with improved economicproperties.

The cell with the target gene may be derived from or contained in anyorganism. The organism may be a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry, and those that are pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies.

Plants include arabidopsis; field crops (e.g., alfalfa, barley, bean,com, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean,sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet,broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant,lettuce, onion, pepper, potato, pumpkin, radish, spinach, squash, taro,tomato, and zucchini); fruit and nut crops (e.g., almond, apple,apricot, banana, blackberry, blueberry, cacao, cherry, coconut,cranberry, date, faJoa, filbert, grape, grapefruit, guava, kiwi, lemon,lime, mango, melon, nectarine, orange, papaya, passion fruit, peach,peanut, pear, pineapple, pistachio, plum, raspberry, strawberry,tangerine, walnut, and watermelon); and ornamentals (e.g., alder, ash,aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,rhododendron, rose, and rubber).

Examples of vertebrate animals include fish, mammal, cattle, goat, pig,sheep, rodent, hamster, mouse, rat, primate, and human.

Invertebrate animals include nematodes, other worms, Drosophila, andother insects. Representative generae of nematodes include those thatinfect animals (e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum,Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus,Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia,Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris,Trichostrongylus, Tflichonema, Toxocara, Uncinaria) and those thatinfect plants (e.g., Bursaphalenchus, Criconerriella, Diiylenchus,Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus,Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus,Rotelynchus, Tylenchus, and Xiphinerna). Representative orders ofinsects include Coleoptera, Diptera, Lepidoptera, and Homoptera.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

C. Targeted Genes

The target gene may be a gene derived from the cell, an endogenous gene,a transgene, or a gene of a pathogen which is present in the cell afterinfection thereof. Depending on the particular target gene and the doseof double stranded RNA material delivered, the procedure may providepartial or complete loss of function for the target gene. Lower doses ofinjected material and longer times after administration of dsRNA mayresult in inhibition in a smaller fraction of cells. Quantitation ofgene expression in a cell may show similar amounts of inhibition at thelevel of accumulation of target mRNA or translation of target protein.

“Inhibition of gene expression” refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a targetgene. “Specificity” refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism (as presented below in the examples) or bybiochemical techniques such as RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS). ForRNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxy acid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of dsRNA may result in inhibition in asmaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or95% of targeted cells). Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell: mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

As disclosed herein, the present invention is not limited to any type oftarget gene or nucleotide sequence. But the following classes ofpossible target genes are listed for illustrative purposes:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,Writ family members, Pax family members, Winged helix family members,Hox family members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2,CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FGR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g.,APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); andenzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hemicellulases, integrases, inulinases, invertases, isomerases, kinases,lactases, lipases, lipoxygenases, lysozymes, nopaline synthases,octopine synthases, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, phytases, plant growth regulatorsynthases, polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases).

D. dsRNA Constructs

The dsRNA construct may comprise one or more strands of polymerizedribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general panic response in some organisms which is generatedby dsRNA. Likewise, bases may be modified to block the activity ofadenosine deaminase. The dsRNA construct may be produced enzymaticallyor by partial/total organic synthesis, any modified ribonucleotide canbe introduced by in vitro enzymatic or organic synthesis.

The dsRNA construct may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleicacids include injection of an RNA solution directly into the cell orextracellular injection into the organism.

The double-stranded structure may be formed by a singleself-complementary RNA strand (such as in the form of shRNA) or twocomplementary RNA strands (such as in the form of siRNA). RNA duplexformation may be initiated either inside or outside the cell. The RNAmay be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

dsRNA constructs containing a nucleotide sequences identical to aportion, of either coding or non-coding sequence, of the target gene arepreferred for inhibition. RNA sequences with insertions, deletions, andsingle point mutations relative to the target sequence (ds RNA similarto the target gene) have also been found to be effective for inhibition.Thus, sequence identity may be optimized by sequence comparison andalignment algorithms known in the art (see Gribskov and Devereux,Sequence Analysis Primer, Stockton Press, 1991, and references citedtherein) and calculating the percent difference between the nucleotidesequences by, for example, the Smith-Waterman algorithm as implementedin the BESTFIT software program using default parameters (e.g.,University of Wisconsin Genetic Computing Group). Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). In certain preferred embodiments, the length of the dsRNA isat least 20, 21 or 22 nucleotides in length, e.g., corresponding in sizeto RNA products produced by Dicer-dependent cleavage. In certainembodiments, the dsRNA construct is at least 25, 50, 100, 200, 300 or400 bases. In certain embodiments, the dsRNA construct is 400-800 basesin length.

In one embodiment, the dsRNA is a single-stranded hairpin ribonucleicacid (shRNA) comprising self complementary sequences of 19 to 100nucleotides that form a duplex region, which self complementarysequences hybridize under intracellular conditions to a target gene,wherein said hairpin RNA: (i) is a substrate for cleavage by a RNaseIIIenzyme to produce a double-stranded RNA product, (ii) does not produce ageneral sequence-independent killing of the mammalian cells, and (iii)reduces expression of said target gene in a manner dependent on thesequence of said complementary regions. In a preferred embodiment, theshRNA comprises a 3′ overhang of about 1-4 nucleotides.

In a related embodiment, he dsRNA is a single-stranded hairpinribonucleic acid (shRNA) comprising self complementary sequences of 19to 100 nucleotides that form a duplex region, which self complementarysequences hybridize under intracellular conditions to a target gene,wherein said hairpin RNA: (i) is cleaved in the mammalian cells toproduce an RNA guide sequence that enters an Argonaut-containingcomplex, (ii) does not produce a general sequence-independent killing ofthe mammalian cells, and (iii) reduces expression of said target gene ina manner dependent on the sequence of said complementary regions. In apreferred embodiment, the shRNA comprises a 3′ overhang of about 1-4nucleotides.

The size of the duplex region of the subject shRNA may be longer (e.g.,anywhere between 19 to about 1000 nucleotides, or 19-about 500 nt, or19-about 250 nt, etc.), but in many applications, about 29 nucleotidesis sufficient. In certain embodiments, the duplex region is any wherebetween about 25-29 nt. In other embodiments, the duplex region is anywhere between about 19-25 nt.

The size of the 3′ overhang may be 1-5 nucleotides, preferably 2-4nucleotides. In one embodiment, the 3′ overhang is 2 nucleotides. Thespecific sequences of the 3′ overhang nucleotides are less important. Inone embodiment, the overhang nucleotides can be any nucleotides,including “non-standard” or modified nucleotides. In other embodiments,the overhang sequences are mostly pyramidines, such as U, C, or T. Inone embodiment, the 2-nucleotide ovehang is UU.

In certain embodiments, the 5′ of the shRNA may have 1-5 nt overhangthat does not pair with the 3′ overhang.

The size of the “loop” between the paired duplex region may vary, butpreferably contains at least about 3-8 nucleotides, such as 4nucleotides.

100% sequence identity between the RNA and the target gene is notrequired to practice the present invention. Thus the invention has theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence.

The dsRNA construct may be synthesized either in vitro or in vitro.Endogenous RNA polymerase of the cell may mediate transcription invitro, or cloned RNA polymerase can be used for transcription in vitroor in vitro. For transcription from a transgene in vitro or anexpression construct, a regulatory region (e.g., promoter, enhancer,silencer, splice donor and acceptor, polyadenylation) may be used totranscribe the dsRNA strand (or strands). Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus. The dsRNA construct may bechemically or enzymatically synthesized by manual or automatedreactions. The dsRNA construct may be synthesized by a cellular RNApolymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). Theuse and production of an expression construct are known in the art (seealso WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135,5,789,214, and 5,804,693; and the references cited therein). Ifsynthesized chemically or by in vitro enzymatic synthesis, the RNA maybe purified prior to introduction into the cell. For example, RNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography or a combination thereof.Alternatively, the dsRNA construct may be used with no or a minimum ofpurification to avoid losses due to sample processing. The dsRNAconstruct may be dried for storage or dissolved in an aqueous solution.The solution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

Physical methods of introducing nucleic acids include injection of asolution containing the dsRNA construct, bombardment by particlescovered by the dsRNA construct, soaking the cell or organism in asolution of the RNA, microinjected into the target (e.g., mammaliantarget) cells, or electroporation of cell membranes in the presence ofthe dsRNA construct. A viral construct packaged into a viral particlewould accomplish both efficient introduction of an expression constructinto the cell and transcription of dsRNA construct encoded by theexpression construct. In one embodiment, the shRNA is a transcriptionalproduct that is transcribed from an expression construct introduced intothe target (e.g., mammalian target) cells, which expression constructcomprises a coding sequence for transcribing said shRNA, operably linkedto one or more transcriptional regulatory sequences. Suchtranscriptional regulatory sequences may include a promoter for an RNApolymerase, such as a cellular RNA polymerase. Examplery but notlimiting promoters include: a U6 promoter, a T7 promoter, a T3 promoter,or an SP6 promoter. In certain embodiments, the transcriptionalregulatory sequences includes an inducible promoter.

The dsRNA constructs may be integrated into the host genome, such thatthe target cells are stably transfected with the dsRNA expressionconstructs. The constructs may be suitable for stable integration intoeither cells in culture or in an animal. For example, the constructs maybe integrated into embryonic cells, such as a mouse ES cell, to generatea transgenic animal. The constructs may also be integrated into adultsomatic cells, either primary cell or established cell line.

In certain embodiments, the expression of a target gene (eitherendogenous or heterologous gene) is attenuated by at least about 33%, orabout 50%, about 60%, 70%, 80%, 90%, 95%, or 99% or more, relative toexpression in cells not treated with the dsRNA (e.g., shRNA).

The shRNA may be chemically synthesized, or in vitro transcripted, andmay further include one or more modifications to phosphate-sugarbackbone or nucleosides residues.

Other methods known in the art for introducing nucleic acids to cellsmay be used, such as lipid-mediated carrier transport, chemical mediatedtransport, such as calcium phosphate, and the like. Thus the dsRNAconstruct may be introduced along with components that perform one ormore of the following activities: enhance RNA uptake by the cell,promote annealing of the duplex strands, stabilize the annealed strands,or other-wise increase inhibition of the target gene.

E. Illustrative Uses

One utility of the present invention is as a method of identifying genefunction in an organism, especially higher eukaryotes, comprising theuse of double-stranded RNA to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the invention to reduce the amount and/or alter thetiming of target gene activity. The invention could be used indetermining potential targets for pharmaceuticals, understanding normaland pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleotide sequence informationfrom genomic and expressed gene sources, including total sequences formammalian genomes, can be coupled with the invention to determine genefunction in a cell or in a whole organism. The preference of differentorganisms to use particular codons, searching sequence databases forrelated gene products, correlating the linkage map of genetic traitswith the physical map from which the nucleotide sequences are derived,and artificial intelligence methods may be used to define putative openreading frames from the nucleotide sequences acquired in such sequencingprojects.

A simple assay would be to inhibit gene expression according to thepartial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

The ease with which the dsRNA construct can be introduced into an intactcell/organism containing the target gene allows the present invention tobe used in high throughput screening (HTS). For example, duplex RNA canbe produced by an amplification reaction using primers flanking theinserts of any gene library derived from the target cell or organism.Inserts may be derived from genomic DNA or mRNA (e.g., cDNA and cRNA).Individual clones from the library can be replicated and then isolatedin separate reactions, but preferably the library is maintained inindividual reaction vessels (e.g., a 96 well microtiter plate) tominimize the number of steps required to practice the invention and toallow automation of the process.

In an exemplary embodiment, the subject invention provides an arrayedlibrary of RNAi constructs. The array may be in the form of solutions,such as multi-well plates, or may be “printed” on solid substrates uponwhich cells can be grown. To illustrate, solutions containing duplexRNAs that are capable of inhibiting the different expressed genes can beplaced into individual wells positioned on a microtiter plate as anordered array, and intact cells/organisms in each well can be assayedfor any changes or modifications in behavior or development due toinhibition of target gene activity.

In one embodiment, the subject method uses an arrayed library of RNAiconstructs to screen for combinations of RNAi that are lethal to hostcells. Synthetic lethality is a bedrock principle of experimentalgenetics. A synthetic lethality describes the properties of twomutations which, individually, are tolerated by the organism but which,in combination, are lethal. The subject arrays can be used to identifyloss-of-function mutations that are lethal in combination withalterations in other genes, such as activated oncogenes orloss-of-function mutations to tumor suppressors. To achieve this, onecan create “phenotype arrays” using cultured cells. Expression of eachof a set of genes, such as the host cell's genome, can be individuallysystematically disrupted using RNA interference. Combination withalterations in oncogene and tumor suppressor pathways can be used toidentify synthetic lethal interactions that may identify noveltherapeutic targets.

In certain embodiments, the RNAi constructs can be fed directly to, orinjected into, the cell/organism containing the target gene.Alternatively, the duplex RNA can be produced by in vitro or in vitrotranscription from an expression construct used to produce the library.The construct can be replicated as individual clones of the library andtranscribed to produce the RNA; each clone can then be fed to, injectedinto, or delivered by another method known in the art to, thecell/organism containing the target gene. The function of the targetgene can be assayed from the effects it has on the cell/organism whengene activity is inhibited. This screening could be amenable to smallsubjects that can be processed in large number, for example, tissueculture cells derived from mammals, especially primates, and mostpreferably humans.

If a characteristic of an organism is determined to be geneticallylinked to a polymorphism through RFLP or QTL analysis, the presentinvention can be used to gain insight regarding whether that geneticpolymorphism might be directly responsible for the characteristic. Forexample, a fragment defining the genetic polymorphism or sequences inthe vicinity of such a genetic polymorphism can be amplified to producean RNA, the duplex RNA can be introduced to the organism or cell, andwhether an alteration in the characteristic is correlated withinhibition can be determined. Of course, there may be trivialexplanations for negative results with this type of assay, for example:inhibition of the target gene causes lethality, inhibition of the targetgene may not result in any observable alteration, the fragment containsnucleotide sequences that are not capable of inhibiting the target gene,or the target gene's activity is redundant.

The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or only in specificcellular compartments or tissues. The functional equivalent ofconditional mutations may be produced by inhibiting activity of thetarget gene when or where it is not required for viability. Theinvention allows addition of RNA at specific times of development andlocations in the organism without introducing permanent mutations intothe target genome.

The present invention may be useful in allowing the inhibition of genesthat have been difficult to inhibit using other methods due to generedundancy. Since the present methods may be used to deliver more thanone dsRNA to a cell or organism, dsRNA identical or similar to more thanone gene, wherein the genes have a redundant function during normaldevelopment, may be delivered.

If alternative splicing produced a family of transcripts that weredistinguished by usage of characteristic exons, the present inventioncan target inhibition through the appropriate exons to specificallyinhibit or to distinguish among the functions of family members. Forexample, a protein factor that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone. That is, thesubject method can be used for selected ablation of splicing variants.

The present invention may be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vitro introduction of RNA to test samples or subjects. Preferredcomponents are the dsRNA and a vehicle that promotes introduction of thedsRNA. Such a kit may also include instructions to allow a user of thekit to practice the invention.

Alternatively, an organism may be engineered to produce dsRNA whichproduces commercially or medically beneficial results, for example,resistance to a pathogen or its pathogenic effects, improved growth, ornovel developmental patterns.

Another aspect of the invention provides a method for attenuatingexpression of a target gene in mammalian cells, comprising introducinginto the mammalian cells a single-stranded hairpin ribonucleic acid(shRNA) comprising self complementary sequences of 19 to 100 nucleotidesthat form a duplex region, which self complementary sequences hybridizeunder intracellular conditions to a target gene, wherein said hairpinRNA: (i) is a substrate for cleavage by a RNaseIII enzyme to produce adouble-stranded RNA product, (ii) does not produce a generalsequence-independent killing of the mammalian cells, and (iii) reducesexpression of said target gene in a manner dependent on the sequence ofsaid complementary regions. In a preferred embodiment, the shRNAcomprises a 3′ overhang of about 1-4 nucleotides.

In a related aspect, the invention provides a method for attenuatingexpression of a target gene in mammalian cells, comprising introducinginto the mammalian cells a single-stranded hairpin ribonucleic acid(shRNA) comprising self complementary sequences of 19 to 100 nucleotidesthat form a duplex region, which self complementary sequences hybridizeunder intracellular conditions to a target gene, wherein said hairpinRNA: (i) is cleaved in the mammalian cells to produce an RNA guidesequence that enters an Argonaut-containing complex, (ii) does notproduce a general sequence-independent killing of the mammalian cells,and (iii) reduces expression of said target gene in a manner dependenton the sequence of said complementary regions. In a preferred emodiment,the shRNA comprises a 3′ overhang of about 1-4 nucleotides.

In yet another embodiment, the invention provides a method forattenuating expression of one or more target genes in mammalian cells,comprising introducing into the mammalian cells a variegated library ofsingle-stranded hairpin ribonucleic acid (shRNA) species, each shRNAspecies comprising self complementary sequences of 19 to 100 nucleotidesthat form duplex regions and which hybridize under intracellularconditions to a target gene, wherein each of said hairpin RNA species:(i) is a substrate for cleavage by a RNaseIII enzyme to produce adouble-stranded RNA product, (ii) does not produce a generalsequence-independent killing of the mammalian cells, and (iii) ifcomplementary to a target sequence, reduces expression of said targetgene in a manner dependent on the sequence of said complementaryregions. In a preferred embodiment, the shRNA comprises a 3′ overhang ofabout 1-4 nucleotides.

In certain embodiments, the variegated library of shRNA species arearrayed a solid substrate.

In another embodiment, the method includes the further step ofidentifying shRNA species of said variegated library which produce adetected phenotype in the mammalian cells.

Yet another aspect of the invention provide a method of enhancing thepotency/activity of an RNAi therapeutic for a mammalian patient, theRNAi therapeutic comprising an siRNA of 19-22 paired polynucleotides,the method comprising replacing the siRNA with a single-stranded hairpinRNA (shRNA) of the subject invention, wherein said duplex regioncomprises the same 19-22 paired polynucleotides of the siRNA. Thisaspect of the invention is partly based on the surprising discovery thatshRNA constructs designed as Dicer substrates perform at least as wellas, and in most cases much better/potent than the corresponding siRNAform of dsRNA (e.g., with the same eventual target guide sequence ofabout 22 nucleotides).

In certain embodiments, the half-maximum inhibition by the RNAitherapeutic is achieved by a concentration of the shRNA at least about20%, or about 30%, 40%, 50%, 60%, 70%, 80%, 90% lower than that of thecorresponding siRNA.

In another embodiment, the end-point inhibition by the shRNA is at leastabout 40%, or about 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, or 10-fold higher than that of the siRNA.

Another aspect of the invention provides a method of designing a shorthairpin RNA (shRNA) construct for RNAi, the shRNA comprising a 3′overhang of about 1-4 nucleotides, the method comprising selecting thenucleotide about 21 bases 5′ to the most 3′-end nucleotide as the firstpaired nucleotide in a cognate doubled-stranded siRNA with the same 3′overhang. Such shRNA can be used, for example, for RNAi in mammaliancells.

In one embodiment, the shRNA comprises about 15-45, preferably about25-29 paired polynucleotides.

In one embodiment, the 3′ overhang has 2 nucleotides.

In one embodiment, the shRNA, when cut by a Dicer enzyme (e.g., a humanDicer enzyme), produces a product siRNA that is either identical to, ordiffer by a single basepair immediately 5′ to the 3′ overhang from thecognate siRNA.

In one embodiment, the shRNA construct has substantially the sameprofiles of off-target gene inhibition effects as compared to thecognate siRNA construct with substantially identical target sequences.

IV. EXEMPLIFICATION

The invention, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

EXAMPLE 1 An RNA-Directed Nuclease Mediates RNAi Gene Silencing

In a diverse group of organisms that includes Caenorhabditis elegans,Drosophila, planaria, hydra, trypanosomes, fungi and plants, theintroduction of double-stranded RNAs inhibits gene expression in asequence-specific manner (Sharp, Genes and Development 13: 139-141,1999; Sanchez-Alvarado and Newmark, PNAS 96: 5049-5054, 1999; Lohman etal., Developmental Biology 214: 211-214, 1999; Cogoni and Macino, Nature399: 166-169, 1999; Waterhouse et al., PNAS 95: 13959-13964, 1998;Montgomery and Fire, Trends Genet. 14: 225-228, 1998; Ngo et al., PNAS95: 14687-14692, 1998). These responses, called RNA interference orpost-transcriptional gene silencing, may provide anti-viral defense,modulate transposition or regulate gene expression (Sharp, Genes andDevelopment 13: 139-141, 1999; Montgomery and Fire, Trends Genet. 14:225-228, 1998; Tabara et al., Cell 99: 123-132, 1999; Ketting et al.,Cell 99: 133-141, 1999; Ratcliff et al., Science 276: 1558-1560, 1997).We have taken a biochemical approach towards elucidating the mechanismsunderlying this genetic phenomenon. Here we show that ‘loss-of-function’phenotypes can be created in cultured Drosophila cells by transfectionwith specific double-stranded RNAs. This coincides with a markedreduction in the level of cognate cellular messenger RNAs. Extracts oftransfected cells contain a nuclease activity that specifically degradesexogenous transcripts homologous to transfected double-stranded RNA.This enzyme contains an essential RNA component. After partialpurification, the sequence-specific nuclease co-fractionates with adiscrete, ˜25-nucleotide RNA species which may confer specificity to theenzyme through homology to the substrate mRNAs.

Although double-stranded RNAs (dsRNAs) can provoke gene silencing innumerous biological contexts including Drosophila (Kennerdell et al.,Cell 95: 1017-1026, 1998; Misquitta and Paterson, PNAS 96: 1451-1456,1999), the mechanisms underlying this phenomenon have remained mostlyunknown. We therefore wanted to establish a biochemically tractablemodel in which such mechanisms could be investigated.

Transient transfection of cultured, Drosophila S2 cells with a lacZexpression vector resulted in β-galactosidase activity that was easilydetectable by an in situ assay (FIG. 1 a). This activity was greatlyreduced by co-transfection with a dsRNA corresponding to the first 300nucleotides of the lacZ sequence, whereas co-transfection with a controldsRNA (CD8) (FIG. 1 a) or with single-stranded RNAs of either sense orantisense orientation (data not shown) had little or no effect. Thisindicated that dsRNAs could interfere, in a sequence-specific fashion,with gene expression in cultured cells.

To determine whether RNA interference (RNAi) could be used to targetendogenous genes, we transfected S2 cells with a dsRNA corresponding tothe first 540 nucleotides of Drosophila cyclin E, a gene that isessential for progression into S phase of the cell cycle. Duringlog-phase growth, untreated S2 cells reside primarily in G2/M (FIG. 1b). Transfection with lacZ dsRNA had no effect on cell-cycledistribution, but transfection with the cyclin E dsRNA caused a G1-phasecell-cycle arrest (FIG. 1 b). The ability of cyclin E dsRNA to provokethis response was length-dependent. Double-stranded RNAs of 540 and 400nucleotides were quite effective, whereas dsRNAs of 200 and 300nucleotides were less potent. Double-stranded cyclin E RNAs of 50 or 100nucleotides were inert in our assay, and transfection with asingle-stranded, antisense cyclin E RNA had virtually no effect.

One hallmark of RNAi is a reduction in the level of mRNAs that arehomologous to the dsRNA. Cells transfected with the cyclin E dsRNA (bulkpopulation) showed diminished endogenous cyclin E mRNA as compared withcontrol cells (FIG. 1 c). Similarly, transfection of cells with dsRNAshomologous to fizzy, a component of the anaphase-promoting complex (APC)or cyclin A, a cyclin that acts in S, G2 and M, also caused reduction oftheir cognate mRNAs (FIG. 1 c). The modest reduction in fizzy mRNAlevels in cells transfected with cyclin A dsRNA probably resulted fromarrest at a point in the division cycle at which fizzy transcription islow (Wolf and Jackson, Current Biology 8: R637-R639, 1998; Kramer etal., Current Biology 8: 1207-1210, 1998). These results indicate thatRNAi may be a generally applicable method for probing gene function incultured Drosophila cells.

The decrease in mRNA levels observed upon transfection of specificdsRNAs into Drosophila cells could be explained by effects attranscriptional or post-transcriptional levels. Data from other systemshave indicated that some elements of the dsRNA response may affect mRNAdirectly (reviewed in Sharp, Genes and Development 13: 139-141, 1999;Montgomery and Fire, Trends Genet. 14: 225-228, 1998). We thereforesought to develop a cell-free assay that reflected, at least in part,RNAi.

S2 cells were transfected with dsRNAs corresponding to either cyclin Eor lacZ. Cellular extracts were incubated with synthetic mRNAs of lacZor cyclin E. Extracts prepared from cells transfected with the540-nucleotide cyclin E dsRNA efficiently degraded the cyclin Etranscript; however, the lacZ transcript was stable in these lysates(FIG. 2 a). Conversely, lysates from cells transfected with the lacZdsRNA degraded the lacZ transcript but left the cyclin E mRNA intact.These results indicate that RNAi ablates target mRNAs through thegeneration of a sequence-specific nuclease activity. We have termed thisenzyme RISC (RNA-induced silencing complex). Although we occasionallyobserved possible intermediates in the degradation process (see FIG. 2),the absence of stable cleavage end-products indicates an exonuclease(perhaps coupled to an endonuclease). However, it is possible that theRNAi nuclease makes an initial endonucleolytic cut and that non-specificexonucleases in the extract complete the degradation process(Shuttleworth and Colman, EMBO J. 7: 427-434, 1988). In addition, ourability to create an extract that targets lacZ in vitro indicates thatthe presence of an endogenous gene is not required for the RNAiresponse.

To examine the substrate requirements for the dsRNA-induced,sequence-specific nuclease activity, we incubated a variety ofcyclin-E-derived transcripts with an extract derived from cells that hadbeen transfected with the 540-nucleotide cyclin E dsRNA (FIG. 2 b, c).Just as a length requirement was observed for the transfected dsRNA, theRNAi nuclease activity showed a dependence on the size of the RNAsubstrate. Both a 600-nucleotide transcript that extends slightly beyondthe targeted region (FIG. 2 b) and an ˜1-kilobase (kb) transcript thatcontains the entire coding sequence (data not shown) were completelydestroyed by the extract. Surprisingly, shorter substrates were notdegraded as efficiently. Reduced activity was observed against either a300- or a 220-nucleotide transcript, and a 100-nucleotide transcript wasresistant to nuclease in our assay. This was not due solely to positioneffects because ˜100-nucleotide transcripts derived from other portionsof the transfected dsRNA behaved similarly (data not shown). Asexpected, the nuclease activity (or activities) present in the extractcould also recognize the antisense strand of the cyclin E mRNA. Again,substrates that contained a substantial portion of the targeted regionwere degraded efficiently whereas those that contained a shorter stretchof homologous sequence (˜130 nucleotides) were recognized inefficiently(FIG. 2 c, as600). For both the sense and antisense strands, transcriptsthat had no homology with the transfected dsRNA (FIG. 2 b, Eout; FIG. 2c, as300) were not degraded. Although we cannot exclude the possibilitythat nuclease specificity could have migrated beyond the targetedregion, the resistance of transcripts that do not contain homology tothe dsRNA is consistent with data from C. elegans. Double-stranded RNAshomologous to an upstream cistron have little or no effect on a linkeddownstream cistron, despite the fact that unprocessed, polycistronicmRNAs can be readily detected (Tabara et al., Science 282: 430-432,1998; Bosher et al., Genetics 153: 1245-1256, 1999). Furthermore, thenuclease was inactive against a dsRNA identical to that used to provokethe RNAi response in vitro (FIG. 2 b). In the in vitro system, neither a5′ cap nor a poly(A) tail was required, as such transcripts weredegraded as efficiently as uncapped and non-polyadenylated RNAs.

Gene silencing provoked by dsRNA is sequence specific. A plausiblemechanism for determining specificity would be incorporation ofnucleic-acid guide sequences into the complexes that accomplishsilencing (Hamilton and Baulcombe, Science 286: 950-952, 1999). Inaccord with this idea, pre-treatment of extracts with a Ca²⁺-dependentnuclease (micrococcal nuclease) abolished the ability of these extractsto degrade cognate mRNAs (FIG. 3). Activity could not be rescued byaddition of non-specific RNAs such as yeast transfer RNA. Althoughmicrococcal nuclease can degrade both DNA and RNA, treatment of theextract with DNAse I had no effect (FIG. 3). Sequence-specific nucleaseactivity, however, did require protein (data not shown). Together, ourresults support the possibility that the RNAi nuclease is aribonucleoprotein, requiring both RNA and protein components.Biochemical fractionation (see below) is consistent with thesecomponents being associated in extract rather than being assembled onthe target mRNA after its addition.

In plants, the phenomenon of co-suppression has been associated with theexistence of small (˜25-nucleotide) RNAs that correspond to the genethat is being silenced (Hamilton and Baulcombe, Science 286: 950-952,1999). To address the possibility that a similar RNA might exist inDrosophila and guide the sequence-specific nuclease in the choice ofsubstrate, we partially purified our activity through severalfractionation steps. Crude extracts contained both sequence-specificnuclease activity and abundant, heterogeneous RNAs homologous to thetransfected dsRNA (FIGS. 2 and 4 a). The RNAi nuclease fractionated withribosomes in a high-speed centrifugation step. Activity could beextracted by treatment with high salt, and ribosomes could be removed byan additional centrifugation step. Chromatography of soluble nucleaseover an anion-exchange column resulted in a discrete peak of activity(FIG. 4 b, cyclin E). This retained specificity as it was inactiveagainst a heterologous mRNA (FIG. 4 b, lacZ). Active fractions alsocontained an RNA species of 25 nucleotides that is homologous to thecyclin E target (FIG. 4 b, northern). The band observed on northernblots may represent a family of discrete RNAs because it could bedetected with probes specific for both the sense and antisense cyclin Esequences and with probes derived from distinct segments of the dsRNA(data not shown). At present, we cannot determine whether the25-nucleotide RNA is present in the nuclease complex in adouble-stranded or single-stranded form.

RNA interference allows an adaptive defense against both exogenous andendogenous dsRNAs, providing something akin to a dsRNA immune response.Our data, and that of others (Hamilton and Baulcombe, Science 286:950-952, 1999), is consistent with a model in which dsRNAs present in acell are converted, either through processing or replication, into smallspecificity determinants of discrete size in a manner analogous toantigen processing. Our results suggest that the post-transcriptionalcomponent of dsRNA-dependent gene silencing is accomplished by asequence-specific nuclease that incorporates these small RNAs as guidesthat target specific messages based upon sequence recognition. Theidentical size of putative specificity determinants in plants (Hamiltonand Baulcombe, supra) and animals predicts a conservation of both themechanisms and the components of dsRNA-induced, post-transcriptionalgene silencing in diverse organisms. In plants, dsRNAs provoke not onlypost-transcriptional gene silencing but also chromatin remodeling andtranscriptional repression (Jones et al., EMBO J. 17: 6385-6393, 1998;Jones et al., Plant Cell 11: 2291-2301, 1999). It is now critical todetermine whether conservation of gene-silencing mechanisms also existsat the transcriptional level and whether chromatin remodeling can bedirected in a sequence-specific fashion by these same dsRNA-derivedguide sequences.

Methods:

Cell culture and RNA methods S2 cells (Schneider, J. Embryol Exp Morpho27: 353-365, 1972) were cultured at 27° C. in 90% Schneider's insectmedia (Sigma), 10% heat inactivated fetal bovine serum (FBS). Cells weretransfected with dsRNA and plasmid DNA by calcium phosphateco-precipitation (DiNocera and Dawid, PNAS 80: 7095-7098, 1983).Identical results were observed when cells were transfected using lipidreagents (for example, Superfect, Qiagen). For FACS analysis, cells wereadditionally transfected with a vector that directs expression of agreen fluorescent protein (GFP)-US9 fusion protein (Kalejta et al., ExpCell Res. 248: 322-328, 1999). These cells were fixed in 90% ice-coldethanol and stained with propidium iodide at 25 μg/ml. FACS wasperformed on an Elite flow cytometer (Coulter). For northern blotting,equal loading was ensured by over-probing blots with a controlcomplementary DNA (RP49). For the production of dsRNA, transcriptiontemplates were generated by polymerase chain reaction such that theycontained T7 promoter sequences on each end of the template. RNA wasprepared using the RiboMax kit (Promega). Confirmation that RNAs weredouble stranded came from their complete sensitivity to RNAse III.Target mRNA transcripts were synthesized using the Riboprobe kit(Promega) and were gel purified before use.

Extract preparation Log-phase S2 cells were plated on 15-cm tissueculture dishes and transfected with 30 μg dsRNA and 30 μg carrierplasmid DNA. Seventy-two hours after transfection, cells were harvestedin PBS containing 5 mM EGTA, washed twice in PBS and once in hypotonicbuffer (10 mM HEPES pH 7.3, 6 mM β-mercaptoethanol). Cells weresuspended in 0.7 packed-cell volumes of hypotonic buffer containingComplete protease inhibitors (Boehringer) and 0.5 units/ml of RNasin(Promega). Cells were disrupted in a dounce homogenizer with a type Bpestle, and lysates were centrifuged at 30,000 g for 20 min.Supernatants were used in an in vitro assay containing 20 mM HEPES pH7.3, 110 mM KOAc, 1 mM Mg(OAc)₂, 3 mM EGTA, 2 mM CaCl₂, 1 mM DTT.Typically, 5 μl extract was used in a 10 μl assay that contained also10,000 c.p.m. synthetic mRNA substrate.

Extract fractionation Extracts were centrifuged at 200,000 g for 3 h andthe resulting pellet (containing ribosomes) was extracted in hypotonicbuffer containing also 1 mM MgCl₂ and 300 mM KOAc. The extractedmaterial was spun at 100,000 g for 1 h and the resulting supernatant wasfractionated on Source 15Q column (Pharmacia) using a KCl gradient inbuffer A (20 mM HEPES pH 7.0, 1 mM dithiothreitol, 1 mM MgCl₂).Fractions were assayed for nuclease activity as described above. Fornorthern blotting, fractions were proteinase K/SDS treated, phenolextracted, and resolved on 15% acrylamide 8M urea gels. RNA waselectroblotted onto Hybond N+ and probed with strand-specific riboprobesderived from cyclin E mRNA. Hybridization was carried out in 500 mMNaPO₄ pH 7.0, 15% formamide, 7% SDS, 1% BSA. Blots were washed in 1×SSCat 37-45° C.

EXAMPLE 2 Role for a Bidentate Ribonuclease in the Initiation Step ofRNA Interference

Genetic approaches in worms, fungi and plants have identified a group ofproteins that are essential for double-stranded RNA-induced genesilencing. Among these are ARGONAUTE family members (e.g. RDE1, QDE2)(Tabara et al., Cell 99: 123-132, 1999; Catalanotto et al., Nature 404:245, 2000; Fagard et al., PNAS 97: 11650-11654, 2000), recQ-familyhelicases (MUT-7, QDE3) (Ketting et al., Cell 99: 133-141, 1999; Cogoniand Macino, Science 286: 2342-2344, 1999), and RNA-dependent RNApolymerases (e.g., EGO-1, QDE1, SGS2/SDE1) (Cogoni and Macino, Nature399: 166-169, 1999; Smardon et al., Current Biology 10: 169-178, 2000;Mourrain et al., Cell 101: 533-542, 2000; Dalmay et al., Cell 101:543-553, 2000). While potential roles have been proposed, none of thesegenes has been assigned a definitive function in the silencing process.Biochemical studies have suggested that PTGS is accomplished by amulticomponent nuclease that targets mRNAs for degradation (Hammond etal., Nature 404: 293-296, 2000; Zamore et al., Cell 101: 25-33, 2000;Tuschl et al., Genes and Development 13: 3191-3197, 1999). We have shownthat the specificity of this complex may derive from the incorporationof a small guide sequence that is homologous to the mRNA substrate(Hammond et al., Nature 404: 293-296, 2000). Originally identified inplants that were actively silencing transgenes (Hamilton and Baulcombe,Science 286: 950-952, 1999), these ˜22 nt. RNAs have been producedduring RNAi in vitro using an extract prepared from Drosophila embryos(Zamore et al., Cell 101: 25-33, 2000). Putative guide RNAs can also beproduced in extracts from Drosophila S2 cells (FIG. 5 a). With the goalof understanding the mechanism of post-transcriptional gene silencing,we have undertaken both biochemical fractionation and candidate geneapproaches to identify the enzymes that execute each step of RNAi.

Our previous studies resulted in the partial purification of a nuclease,RISC, that is an effector of RNA interference. See Example 1. Thisenzyme was isolated from Drosophila S2 cells in which RNAi had beeninitiated in vitro by transfection with dsRNA. We first sought todetermine whether the RISC enzyme and the enzyme that initiates RNAi viaprocessing of dsRNA into 22 mers are distinct activities. RISC activitycould be largely cleared from extracts by high-speed centrifugation(100,000×g for 60 min.) while the activity that produces 22 mersremained in the supernatant (FIG. 5 b,c). This simple fractionationindicated that RISC and the 22 mer-generating activity are separable andthus distinct enzymes. However, it seems likely that they might interactat some point during the silencing process.

RNAse III family members are among the few nucleases that showspecificity for double-stranded RNA (Nicholson, FEMS Microbiol Rev 23:371-390, 1999). Analysis of the Drosophila and C. elegans genomesreveals several types of RNAse III enzymes. First is the canonical RNAseIII which contains a single RNAse III signature motif and adouble-stranded RNA binding domain (dsRBD; e.g. RNC_CAEEL). Second is aclass represented by Drosha (Filippov et al., Gene 245: 213-221, 2000),a Drosophila enzyme that contains two RNAse III motifs and a dsRBD(CeDrosha in C. elegans). A third class contains two RNAse IIIsignatures and an amino terminal helicase domain (e.g. DrosophilaCG4792, CG6493, C. elegans K12H4.8), and these had previously beenproposed by Bass as candidate RNAi nucleases (Bass, Cell 101: 235-238,2000). Representatives of all three classes were tested for the abilityto produce discrete, ˜22 nt. RNAs from dsRNA substrates.

Partial digestion of a 500 nt. cyclin E dsRNA with purified, bacterialRNAse III produced a smear of products while nearly complete digestionproduced a heterogeneous group of ˜11-17 nucleotide RNAs (not shown). Inorder to test the dual-RNAse III enzymes, we prepared T7 epitope-taggedversions of Drosha and CG4792. These were expressed in transfected S2cells and isolated by immunoprecipitation using antibody-agaroseconjugates. Treatment of the dsRNA with the CG4792 immunoprecipitateyielded ˜22 nt. fragments similar to those produced in either S2 orembryo extracts (FIG. 6 a). Neither activity in extract nor activity inimmunoprecipitates depended on the sequence of the RNA substrate sincedsRNAs derived from several genes were processed equivalently (seeSupplement 1). Negative results were obtained with Drosha and withimmunoprecipitates of a DExH box helicase (Homeless (Gillespie et al.,Genes and Development 9: 2495-2508, 1995); see FIG. 6 a,b). Westernblotting confirmed that each of the tagged proteins was expressed andimmunoprecipitated similarly (see Supplement 2). Thus, we conclude thatCG4792 may carry out the initiation step of RNA interference byproducing ˜22 nt. guide sequences from dsRNAs. Because of its ability todigest dsRNA into uniformly sized, small RNAs, we have named this enzymeDicer (Dcr). Dicer mRNA is expressed in embryos, in S2 cells, and inadult flies, consistent with the presence of functional RNAi machineryin all of these contexts (see Supplement 3).

The possibility that Dicer might be the nuclease responsible for theproduction of guide RNAs from dsRNAs prompted us to raise an antiserumdirected against the carboxy-terminus of the Dicer protein (Dicer-1,CG4792). This antiserum could immunoprecipitate a nuclease activity fromeither Drosophila embryo extracts or from S2 cell lysates that produced˜22 nt. RNAs from dsRNA substrates (FIG. 6C). The putative guide RNAsthat are produced by the Dicer-1 enzyme precisely co-migrate with 22mers that are produced in extract and with 22 mers that are associatedwith the RISC enzyme (FIG. 6 D,F). It had previously been shown that theenzyme that produced guide RNAs in Drosophila embryo extracts wasATP-dependent (Zamore et al., Cell 101: 25-33, 2000). Depletion of thiscofactor resulted in an ˜6-fold lower rate of dsRNA cleavage and in theproduction of RNAs with a slightly lower mobility. Of interest was thefact that both Dicer-1 immunoprecipitates and extracts from S2 cellsrequire ATP for the production of ˜22 mers (FIG. 6D). We do not observethe accumulation of lower mobility products in these cases, although wedo routinely observe these in ATP-depleted embryo extracts. Therequirement of this nuclease for ATP is a quite unusual property. Wehypothesize that this requirement could indicate that the enzyme may actprocessively on the dsRNA, with the helicase domain harnessing theenergy of ATP hydrolysis both for unwinding guide RNAs and fortranslocation along the substrate.

Efficient induction of RNA interference in C. elegans and in Drosophilahas several requirements. For example, the initiating RNA must bedouble-stranded, and it must be several hundred nucleotides in length.To determine whether these requirements are dictated by Dicer, wecharacterized the ability of extracts and of immunoprecipitated enzymeto digest various RNA substrates. Dicer was inactive against singlestranded RNAs regardless of length (see Supplement 4). The enzyme coulddigest both 200 and 500 nucleotide dsRNAs but was significantly lessactive with shorter substrates (see Supplement 4). Double-stranded RNAsas short as 35 nucleotides could be cut by the enzyme, albeit veryinefficiently (data not shown). In contrast, E. coli RNAse III coulddigest to completion dsRNAs of 35 or 22 nucleotides (not shown). Thissuggests that the substrate preferences of the Dicer enzyme maycontribute to but not wholly determine the size dependence of RNAi.

To determine whether the Dicer enzyme indeed played a role in RNAi invitro, we sought to deplete Dicer activity from S2 cells and test theeffect on dsRNA-induced gene silencing. Transfection of S2 cells with amixture of dsRNAs homologous to the two Drosophila Dicer genes (CG4792and CG6493) resulted in an ˜6-7 fold reduction of Dicer activity eitherin whole cell lysates or in Dicer-1 immunoprecipitates (FIG. 7A,B).Transfection with a control dsRNA (murine caspase 9) had no effect.Qualitatively similar results were seen if Dicer was examined byNorthern blotting (not shown). Depletion of Dicer in this mannersubstantially compromised the ability of cells to silence subsequentlyan exogenous, GFP transgene by RNAi (FIG. 7C). These results indicatethat Dicer is involved in RNAi in vitro. The lack of complete inhibitionof silencing could result from an incomplete suppression of Dicer (whichis itself required for RNAi) or could indicate that in vitro, guide RNAscan be produced by more than one mechanism (e.g. through the action ofRNA-dependent RNA polymerases).

Our results indicate that the process of RNA interference can be dividedinto at least two distinct steps. According to this model, initiation ofPTGS would occur upon processing of a double-stranded RNA by Dicer into˜22 nucleotide guide sequences, although we cannot formally exclude thepossibility that another, Dicer-associated nuclease may participate inthis process. These guide RNAs would be incorporated into a distinctnuclease complex (RISC) that targets single-stranded mRNAs fordegradation. An implication of this model is that guide sequences arethemselves derived directly from the dsRNA that triggers the response.In accord with this model, we have demonstrated that ³²P-labeled,exogenous dsRNAs that have been introduced into S2 cells by transfectionare incorporated into the RISC enzyme as 22 mers (FIG. 7E). However, wecannot exclude the possibility that RNA-dependent RNA polymerases mightamplify 22 mers once they have been generated or provide an alternativemethod for producing guide RNAs.

The structure of the Dicer enzyme provokes speculation on the mechanismby which the enzyme might produce discretely sized fragmentsirrespective of the sequence of the dsRNA (see Supplement 1, FIG. 8 a).It has been established that bacterial RNAse III acts on its substrateas a dimer (Nicholson, FEMS Microbiol Rev 23: 371-390, 1999; Robertsonet al., J Biol Chem 243: 82-91, 1968; Dunn, J Biol Chem 251: 3807-3814,1976). Similarly, a dimer of Dicer enzymes may be required for cleavageof dsRNAs into ˜22 nt. pieces. According to one model, the cleavageinterval would be determined by the physical arrangement of the twoRNAse III domains within Dicer enzyme (FIG. 8 a). A plausiblealternative model would dictate that cleavage was directed at a singleposition by the two RIII domains in a single Dicer protein. The 22nucleotide interval could be dictated by interaction of neighboringDicer enzymes or by translocation along the mRNA substrate. The presenceof an integral helicase domain suggests that the products of Dicercleavage might be single-stranded 22 mers that are incorporated into theRISC enzyme as such.

A notable feature of the Dicer family is its evolutionary conservation.Homologs are found in C. elegans (K12H4.8), Arabidopsis (e.g., CARPELFACTORY (Jacobson et al., Development 126: 5231-5243, 1999), T25K16.4,AC012328_(—)1), mammals (Helicase-MOI (Matsuda et al., Biochim BiophysActa 1490: 163-169, 2000) and S. pombe (YC9A_SCHPO) (FIG. 8 b, seeSupplements 6,7 for sequence comparisons). In fact, the human Dicerfamily member is capable of generating ˜22 nt. RNAs from dsRNAsubstrates (Supplement 5) suggesting that these structurally similarproteins may all share similar biochemical functions. It has beendemonstrated that exogenous dsRNAs can affect gene function in earlymouse embryos (Wianny et al., Nature Cell Biology 2: 70-75, 2000), andour results suggest that this regulation may be accomplished by anevolutionarily conserved RNAi machinery.

In addition to RNAseIII and helicase motifs, searches of the PFAMdatabase indicate that each Dicer family member also contains a ZAPdomain (FIG. 8 c) (Sonnhammer et al., Proteins 28: 405-420, 1997). Thissequence was defined based solely upon its conservation in theZwille/ARGONAUTE/Piwi family that has been implicated in RNAi bymutations in C. elegans (Rde-1) and Neurospora (Qde-2) (Tabara et al.,Cell 99: 123-132, 1999; Catalanotto et al., Nature 404: 245, 2000).Although the function of this domain is unknown, it is intriguing thatthis region of homology is restricted to two gene families thatparticipate in dsRNA-dependent silencing. Both the ARGONAUTE and Dicerfamilies have also been implicated in common biological processes,namely the determination of stem-cell fates. A hypomorphic allele ofcarpel factory, a member of the Dicer family in Arabidopsis, ischaracterized by increased proliferation in floral meristems (Jacobsenet al., Development 126: 5231-5243, 1999). This phenotype and a numberof other characteristic features are also shared by ArabidopsisARGONAUTE (ago1-1) mutants (Bohmert et al., EMBO J 17: 170-180, 1998; C.Kidner and R. Martiennsen, pers. comm.). These genetic analyses begin toprovide evidence that RNAi may be more than a defensive response tounusual RNAs but may also play important roles in the regulation ofendogenous genes.

With the identification of Dicer as a catalyst of the initiation step ofRNAi, we have begun to unravel the biochemical basis of this unusualmechanism of gene regulation. It will be of critical importance todetermine whether the conserved family members from other organisms,particularly mammals, also play a role in dsRNA-mediated generegulation.

Methods:

Plasmid constructs. A full-length cDNA encoding Drosha was obtained byPCR from an EST sequenced by the Berkeley Drosophila genome project. TheHomeless clone was a gift from Gillespie and Berg (Univ. Washington).The T7 epitope-tag was added to the amino terminus of each by PCR, andthe tagged cDNAs were cloned into pRIP, a retroviral vector designedspecifically for expression in insect cells (E. Bernstein, unpublished).In this vector, expression is driven by the Orgyia pseudotsugata IE2promoter (Invitrogen). Since no cDNA was available for CG4792/Dicer, agenomic clone was amplified from a bacmid (BACR23F10; obtained from theBACPAC Resource Center in the Dept. of Human Genetics at the RoswellPark Cancer Institute). Again, during amplification, a T7 epitope tagwas added at the amino terminus of the coding sequence. The human Dicergene was isolated from a cDNA library prepared from HaCaT cells (GJH,unpublished). A T7-tagged version of the complete coding sequence wascloned into pCDNA3 (Invitrogen) for expression in human cells (LinX-A).

Cell culture and extract preparation. S2 and embryo culture. S2 cellswere cultured at 27° C. in 5% CO₂ in Schneider's insect mediasupplemented with 10% heat inactivated fetal bovine serum (Gemini) and1% antibiotic-antimycotic solution (GIBCO BRL). Cells were harvested forextract preparation at 10×10⁶ cells/ml. The cells were washed 1× in PBSand were resuspended in a hypotonic buffer (10 mM HEPES pH 7.0, 2 mMMgCl₂, 6 mM βME) and dounced. Cell lysates were spun 20,000×g for 20minutes. Extracts were stored at −80° C. Drosophila embryos were rearedin fly cages by standard methodologies and were collected every 12hours. The embryos were dechorionated in 50% chlorox bleach and washedthoroughly with distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl,1.5 mM MgCl₂, 0.5 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 0.2 mMPMSF) was added to the embryos, and extracts were prepared byhomogenization in a tissue grinder. Lysates were spun for two hours at200,000×g and were frozen at −80° C. LinX-A cells, ahighly-transfectable derivative of human 293 cells, (Lin Xie and GJH,unpublished) were maintained in DMEM/10% FCS.

Transfections and immunoprecipitations. S2 cells were transfected usinga calcium phosphate procedure essentially as previously described(Hammond et al., Nature 404: 293-296, 2000). Transfection rates were˜90% as monitored in controls using an in situ β-galactosidase assay.LinX-A cells were also transfected by calcium phosphateco-precipitation. For immunoprecipitations, cells (5×10⁶ per IP) weretransfected with various clones and lysed three days later in IP buffer(125 mM KOAc, 1 mM MgOAc, 1 mM CaCl₂, 5 mM EGTA, 20 mM Hepes pH 7.0, 1mM DTT, 1% NP-40 plus Complete protease inhibitors, Roche). Lysates werespun for 10 minutes at 14,000×g and supernatants were added to T7antibody-agarose beads (Novagen). Antibody binding proceeded for 4 hoursat 4° C. Beads were centrifuged and washed in lysis buffer three times,and once in reaction buffer. The Dicer antiserum was raised in rabbitsusing a KLH-conjugated peptide corresponding to the C-terminal 8 aminoacids of Drosophila Dicer-1 (CG4792).

Cleavage reactions. RNA preparation. Templates to be transcribed intodsRNA were generated by PCR with forward and reverse primers, eachcontaining a T7 promoter sequence. RNAs were produced using Riboprobe(Promega) kits and were uniformly labeling during the transcriptionreaction with ³²P-UTP. Single-stranded RNAs were purified from 1%agarose gels. dsRNA cleavage. Five microliters of embryo or S2 extractswere incubated for one hour at 30° C. with dsRNA in a reactioncontaining 20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5%Superasin (Ambion). Immunoprecipitates were treated similarly exceptthat a minimal volume of reaction buffer (including ATP and Superasin)and dsRNA were added to beads that had been washed in reaction buffer(see above). For ATP depletion, Drosophila embryo extracts wereincubated for 20 minutes at 30° C. with 2 mM glucose and 0.375 U ofhexokinase (Roche) prior to the addition of dsRNA.

Northern and Western analysis. Total RNA was prepared from Drosophilaembryos (0-12 hour), from adult flies, and from S2 cells using Trizol(Lifetech). Messenger RNA was isolated by affinity selection usingmagnetic oligo-dT beads (Dynal). RNAs were electrophoresed on denaturingformaldehyde/agarose gels, blotted and probed with randomly primed DNAscorresponding to Dicer. For Western analysis, T7-tagged proteins wereimmunoprecipitated from whole cell lysates in IP buffer usinganti-T7-antibody-agarose conjugates. Proteins were released from thebeads by boiling in Laemmli buffer and were separated by electrophoresison 8% SDS PAGE. Following transfer to nitrocellulose, proteins werevisualized using an HRP-conjugated anti-T7 antibody (Novagen) andchemiluminescent detection (Supersignal, Pierce).

RNAi of Dicer. Drosophila S2 cells were transfected either with a dsRNAcorresponding to mouse caspase 9 or with a mixture of two dsRNAscorresponding to Drosophila Dicer-1 and Dicer-2 (CG4792 and CG6493). Twodays after the initial transfection, cells were again transfected with amixture containing a GFP expression plasmid and either luciferase dsRNAor GFP dsRNA as previously described (Hammond et al., Nature 404:293-296, 2000). Cells were assayed for Dicer activity or fluorescencethree days after the second transfection. Quantification of fluorescentcells was done on a Coulter EPICS cell sorter after fixation. Controltransfections indicated that Dicer activity was not affected by theintroduction of caspase 9 dsRNA.

EXAMPLE 3 A Simplified Method for the Creation of Hairpin Constructs forRNA Interference

In numerous model organisms, double stranded RNAs have been shown tocause effective and specific suppression of gene function (Bosher andLabouesse, Nature Cell Biology 2: E31-E36, 2000). This response, termedRNA interference or post-transcriptional gene silencing, has evolvedinto a highly effective reverse genetic tool in C. elegans, Drosophila,plants and numerous other systems. In these cases, double-stranded RNAscan be introduced by injection, transfection or feeding; however, in allcases, the response is both transient and systemic. Recently, stableinterference with gene expression has been achieved by expression ofRNAs that form snap-back or hairpin structures (Fortier and Belote,Genesis 26: 240-244, 2000; Kennerdell and Carthew, Nature Biotechnology18: 896-898, 2000; Lam and Thummel, Current Biology 10: 957-963, 2000;Shi et al., RNA 6: 1069-1076, 2000; Smith et al., Nature 407: 319-320,2000; Tavernarakis et al., Nature Genetics 24: 180-183, 2000). This hasthe potential not only to allow stable silencing of gene expression butalso inducible silencing as has been observed in trypanosomes and adultDrosophila (Fortier and Belote, Genesis 26: 240-244, 2000; Lam andThummel, Current Biology 10: 957-963, 2000; Shi et al., RNA 6:1069-1076, 2000). The utility of this approach is somewhat hampered bythe difficulties that arise in the construction of bacterial plasmidscontaining the long inverted repeats that are necessary to provokesilencing. In a recent report, it was stated that more than 1,000putative clones were screened to identify the desired construct(Tavernarakis et al., Nature Genetics 24: 180-183, 2000).

The presence of hairpin structures often induces plasmid rearrangement,in part due to the E. coli sbc proteins that recognize and cleavecruciform DNA structures (Connelly et al., Genes Cell 1: 285-291, 1996).We have developed a method for the construction of hairpins that doesnot require cloning of inverted repeats, per se. Instead, the fragmentof the gene that is to be silenced is cloned as a direct repeat, and theinversion is accomplished by treatment with a site-specific recombinase,either in vitro (or potentially in vitro) (see FIG. 27). Followingrecombination, the inverted repeat structure is stable in a bacterialstrain that lacks an intact SBC system (DL759). We have successfullyused this strategy to construct numerous hairpin expression constructsthat have been successfully used to provoke gene silencing in Drosophilacells.

In the following examples, we use this method to express long dsRNAs ina variety of mammalian cell types. We show that such long dsRNAs mediateRNAi in a variety of cell types. Additionally, since the vectordescribed in FIG. 27 contains a selectable marker, dsRNAs produced inthis manner can be stably expressed in cells. Accordingly, this methodallows not only the examination of transient effects of RNA suppressionin a cell, but also the effects of stable and prolonged RNA suppression.

Methods:

Plasmids expressing hairpin RNAs were constructed by cloning the first500 bps of the GFP coding region into the FLIP cassette of pRIP-FLIP asa direct repeat. The FLIP cassette contains two directional cloningsites, the second of which is flanked by LoxP sites. The Zeocin gene,present between the cloning sites, maintains selection and stability. Tocreate an inverted repeat for hairpin production, the direct repeatclones were exposed to Cre recombinase (Stratagene) in vitro and,afterwards, transformed into DL759 E. coli. These bacteria permit thereplication of DNA containing cruciform structures, which tend to forminverted repeats.

EXAMPLE 4 Long dsRNAs Suppress Gene Expression in Mammalian Cells

Previous experiments have demonstrated that dsRNA, produced using avariety of methods including via the construction of hairpins, cansuppress gene expression in Drosophila cells. We now demonstrate thatdsRNA can also suppress gene expression in mammalian cells in culture.Additionally, the power of RNAi as a genetic tool would be greatlyenhanced by the ability to engineer stable silencing of gene expression.We therefore undertook an effort to identify mammalian cells in whichlong dsRNAs could be used as RNAi triggers in the hope that these samecell lines would provide a platform upon which to develop stablesilencing strategies. We demonstrate that RNA suppression can bemediated by stably expressing a long hairpin in a mammalian cell line.The ability to engineer stable silencing of gene expression in culturedmammalian cells, in addition to the ability to transiently silence geneexpression, has many important applications.

A. RNAi in Pluripotent Murine P19 Cells.

We first sought to determine whether long dsRNA triggers could inducesequence-specific silencing in cultured murine cells, both to developthis approach as a tool for probing gene function and to allowmechanistic studies of dsRNA-induced silencing to be propagated tomammalian systems. We, therefore, attempted to extend previous studiesin mouse embryos (Wianny et al., Nat. Cell Biol. 2: 70-75, 2000; Svobodaet al., Development 127: 4147-4156, 2000) by searching for RNAi-likemechanisms in pluripotent, embryonic cell types. We surveyed a number ofcell lines of embryonic origin for the degree to which generalizedsuppression of gene expression occurred upon introduction of dsRNA. Asan assay, we tested the effects of dsRNA on the expression of GFP asmeasured in situ by counting fluorescent cells. As expected, in bothhuman embryonic kidney cells (293) and mouse embryo fibroblasts, GFPexpression was virtually eliminated irrespective of the sequence of thecotransfected dsRNA. In some pluripotent teratocarcinoma and teratomacell lines (e.g., N-Tera1, F9), the PKR response was attenuated butstill evident; however, in contrast, transfection of nonhomologousdsRNAs had no effect on the expression of reporter genes (e.g., GFP orluciferase) either in mouse embryonic stem cells or in p19 embryonalcarcinoma cells (FIG. 28).

Transfection of P19 embryonal carcinoma cells with GFP in the presenceof cognate dsRNA corresponding to the first ≈500 nts of the GFP codingsequence had a strikingly different effect. GFP expression waseliminated in the vast majority of cotransfected cells (FIG. 28),suggesting that these cultured murine cells might respond to dsRNA in amanner similar to that which we had previously demonstrated in cultured,Drosophila S2 cells (Hammond et al., Nature 404: 293-296, 2000).

To quantify the extent to which dsRNA could induce sequence-specificgene silencing, we used a dual luciferase reporter assay similar to thatwhich had first been used to demonstrate RNAi in Drosophila embryoextracts (Tuschl et al., Genes Dev. 13: 3191-3197, 1999). P19 EC cellswere transfected with a mixture of two plasmids that individually directthe expression of firefly luciferase and Renilla luciferase. These werecotransfected with no dsRNA, with dsRNA that corresponds to the first≈500 nts of the firefly luciferase, or with dsRNA corresponding to thefirst ≈500 nts of GFP as a control. Cotransfection with GFP dsRNA gaveluciferase activities that were similar to the no-dsRNA control, both inthe firefly/Renilla activity ratio and in the absolute values of bothactivities. In contrast, in cells that received the firefly luciferasedsRNA, the ratio of firefly to Renilla luciferase activity was reducedby up to 30-fold (250 ng, FIG. 29B). For comparison, we carried out anidentical set of experiments in Drosophila S2 cells. Althoughqualitatively similar results were obtained, the silencing response wasmore potent. At equivalent levels of dsRNA, S2 cells suppressed fireflyluciferase activity to virtually background levels.

The complementary experiment, in which dsRNA was homologous to Renillaluciferase, was also performed. Again, in this case, suppression of theexpression of the Renilla enzyme was ≈10-fold (FIG. 29D). Thus, thedsRNA response in P19 cells was flexible, and the silencing machinerywas able to adapt to dsRNAs directed against any of the reporters thatwere tested.

We took two approaches to test whether this response was specific fordsRNA. Pretreatment of the trigger with purified RNase III, adsRNA-specific ribonuclease, before transfection greatly reduced itsability to provoke silencing. Furthermore, transfection of cells withsingle-stranded antisense RNAs directed against either firefly orRenilla luciferase had little or no effect on expression of thereporters (FIG. 29C and 29D). Considered together, these resultsprovided a strong indication that double-stranded RNAs provoke a potentand specific silencing response in P19 embryonal carcinoma cells.Efficient silencing could be provoked with relatively low concentrationsof dsRNA (25 ng/ml culture media; see FIG. 29A). The response wasconcentration-dependent, with maximal suppression of ≈20-fold beingachieved at a dose of 1.5 μg/ml culture media. Silencing was establishedrapidly and was evident by 9 h post-transfection (the earliest timepoint examined). Furthermore, the response persisted without significantchanges in the degree of suppression for up to 72 h following a singledose of dsRNA.

FIG. 30 further shows wild-type P19 cells which have been co-transfectedwith either RFP or GFP (right panel). Note the robust expression of RFPor GFR respectively approximately 42 hours post-transfection. Weisolated P19 clones which stably express a 500 nt. GFP hairpin. Suchclones were then transfected with either RFP or GFP, and expression ofRFP or GFP was assessed by visual inspection of the cells. The leftpanel demonstrates that a 500 nt GFP hairpin specifically suppressesexpression of GFP in P19 cells.

B. RNAi in Embryonic Stem Cells.

To assess whether the presence of a sequence-specific response to dsRNAwas a peculiarity of P19 cells or whether it also extended to normalmurine embryonic cells, we performed similar silencing assays in mouseembryonic stem cells. Cotransfection of embryonic stem cells withnoncognate dsRNAs (e.g. GFP), again, had no dramatic effect on eitherthe absolute values or the ratios of Renilla and firefly luciferaseactivity (FIG. 31). However, transfection with either firefly or Renillaluciferase dsRNA dramatically and specifically reduced the activity ofthe targeted enzyme (FIG. 31).

This result suggests that RNAi can operate in multiple murine cell typesof embryonic origin, including normal embryonic stem cells. The abilityto provoke silencing in a cell type that is normally used for thegeneration of genetic, mosaic animals suggests the possibility ofeventually testing the biological effects of silencing both in cultureand in reconstituted animal models. Our ability to successfullymanipulate ES cell via RNAi allows the use of RNAi in the generation oftransgenic and knock-out mice.

C. RNAi in Murine Somatic Cells.

RNAi effector pathways are likely to be present in mammalian somaticcells, based on the ability of siRNAs to induce transient silencing(Elbashir et al., Nature 411: 494-498, 2001). Furthermore, we have shownthat RNAi initiator and effector pathways clearly exist in embryoniccells that can enforce silencing in response to long dsRNA triggers. Wetherefore sought to test whether the RNAi machinery might exist intactin some somatic cell lines.

Transfection of HeLa cells with luciferase reporters in combination withlong dsRNA triggers caused a nearly complete suppression of activity,irrespective of the RNA sequence. In a murine myoblast cell line, C2C12,we noted a mixture of two responses. dsRNAs homologous to fireflyluciferase provoked a sequence-specific effect, producing a degree ofsuppression that was slightly more potent than was observed upontransfection with cognate ≈21-nt siRNA (Elbashir et al., Nature 411:494-498, 2001) (see FIG. 32A). However, with long dsRNA triggers, thespecific effect was superimposed upon a generalized suppression ofreporter gene expression that was presumably because of PKR activation(FIG. 32B).

Numerous mammalian viruses have evolved the ability to block PKR as anaid to efficient infection. For example, adenoviruses express VA RNAs,which mimic dsRNA with respect to binding but not to activation of PKR(Clarke et al., RNA 1: 7-20, 1995). Vaccinia virus uses two strategiesto evade PKR. The first is expression of E3L, which binds and masksdsRNAs (Kawagishi-Kobayashi et al., Virology 276: 424-434, 2000). Thesecond is expression of K3L, which binds and inhibits PKR via itsability to mimic the natural substrate of this enzyme, eIF2α(Kawagishi-Kobayashi et al. 2000, supra).

Transfection of C2C12 cells with a vector that directs K3L expressionattenuates the generalized repression of reporter genes in response todsRNA. However, this protein had no effect on the magnitude of specificinhibition by RNAi (FIG. 32C).

FIG. 33 further shows the results of a transient co-transfection assayperformed in Hela cells, CHO cells, and P19 cells. The cell lines wereeach transfected with plasmids expressing Photinus pyralis (firefly) andRenila reniformis (sea pansy) luciferases. The cells lines wereadditionally transfected with 400 ng of 500 nt dsRNAs corresponding toeither firefly luciferase (dsLUC) or dsGFP. The results demonstrate thatdsRNA can specifically mediate suppression in a multiple mammalian cellstypes in culture.

These results raise the possibility that, at least in some cell linesand/or cell types, blocking nonspecific responses to dsRNA will enablethe use of long dsRNAs for the study of gene function. This might beaccomplished through the use of viral inhibitors, as described here, orthrough the use of cells isolated from animals that are geneticallymodified to lack undesirable responses.

D. Stable Suppression of Gene Expression Using RNAi.

To date, dsRNAs have been used to induce sequence-specific genesilencing in either cultured mammalian cells or in embryos only in atransient fashion. However, the most powerful applications of geneticmanipulation are realized only with the creation of stable mutants. Theability to induce silencing by using long dsRNAs offers the opportunityto translate into mammalian cells work from model systems such asDrosophila, plants, and C. elegans wherein stable silencing has beenachieved by enforced expression of hairpin RNAs (Kennerdell et al., Nat.Biotechnol. 18: 896-898, 2000; Smith et al., Nature 407: 319-320, 2000;Tavernarakis et al., Nat. Genet. 24: 180-183, 2000).

P19 EC cells were transfected with a control vector or with anexpression vector that directs expression of a ≈500-nt GFP hairpin RNAfrom an RNA polymerase II promoter (cytomegalovirus). Colonies arisingfrom cells that had stably integrated either construct were selected andexpanded into clonal cell lines. Each cell line was assayed forpersistent RNAi by transient co-transfection with a mixture of tworeporter genes, dsRED to mark transfected cells and GFP to test forstable silencing.

Transfection of clonal P19 EC cells that had stably integrated thecontrol vector produced equal numbers of red and green cells, as wouldbe expected in the absence of any specific silencing response (FIG.34B), whereas cells that express the GFP hairpin RNA gave a verydifferent result. These cells expressed the dsRED protein with anefficiency comparable to that observed in cells containing the controlvector. However, the cells failed to express the cotransfected GFPreporter (FIG. 34B). These data provide a strong indication thatcontinuous expression of a hairpin dsRNA can provoke stable,sequence-specific silencing of a target gene.

In Drosophila S2 cells and C. elegans, RNAi is initiated by the Dicerenzyme, which processes dsRNA into ≈22-nt siRNAs (Bernstein et al.,Nature 409: 363-366, 2001; Grishok et al., Cell 106: 23-34, 2001;Hutvagner et al., Science 293: 834-838, 2001; Ketting et al., Genes Dev.15: 2654-2659, 2001; Knight et al., Science 293: 2269-2271, 2001). Inboth, S2 cells and C. elegans experiments by using dsRNA to target Dicersuppress the RNAi response. Whether Dicer plays a central role inhairpin-induced gene silencing in P19 cells was tested by transfectingP19 cells stably transfected with GFP hairpin constructs with mouseDicer dsRNA. Treatment with Dicer dsRNA, but not control dsRNA, resultedin derepression of GFP (FIG. 34C).

E. dsRNA Induces Posttranscriptional Silencing.

A key feature of RNAi is that it exerts its effect at theposttranscriptional level by destruction of targeted mRNAs (Hammond etal., Nat. Rev. Genet. 2: 110-119, 2001). To test whether dsRNAs inducedsilencing in mouse cells via posttranscriptional mechanisms, we used anassay identical to that, used initially to characterize RNAi responsesin Drosophila embryo extracts (Tuschl et al., Genes Dev. 13: 3191-3197,1999). We prepared lysates from P19 EC cells that were competent for invitro translation of capped mRNAs corresponding to Renilla and fireflyluciferase. Addition of nonspecific dsRNAs to these extracts had nosubstantial effect on either the absolute amount of luciferaseexpression or on the ratio of firefly to Renilla luciferase (FIG. 35).In contrast, addition of dsRNA homologous to the firefly luciferaseinduced a dramatic and dose-dependent suppression of activity. Additionof RNA corresponding to only the antisense strand of the dsRNA hadlittle effect, comparable to a nonspecific dsRNA control, andpretreatment of the dsRNA silencing trigger with RNase III greatlyreduced its potential to induce silencing in vitro. A second hallmark ofRNAi is the production of small, ≈22-nt siRNAs, which determine thespecificity of silencing. We found that such RNA species were generatedfrom dsRNA in P19 cell extracts (FIG. 34D, in vitro), indicative of thepresence of a mouse Dicer activity. These species were also produced incells that stably express GFP hairpin RNAs (FIG. 34D, in vitro).Considered together, the posttranscriptional nature of dsRNA-inducedsilencing, the association of silencing with the production of ≈22-ntsiRNAs, and the dependence of this response on Dicer, a key player inthe RNAi pathway, strongly suggests that dsRNA suppresses geneexpression in murine cells via a conventional RNAi mechanism.

F. RNAi-Mediated Gene Silencing is Specific and Requires dsRNAs.

We carried out experiments to verify that the suppressive effectsobserved in the in vitro system were specific to double stranded RNA.Briefly, experiments were performed in accordance with the methodsoutlined above. Either dsRNA (ds), single-stranded RNA (ss), orantisense-RNA (as) corresponding to firefly (FF) or Renilla (Ren)luciferase was added to the translation reaction. Following reactionsperformed at 30° C. for 1 hour, dual luciferase assays were performedusing an Analytical Scientific Instruments model 3010 Luminometer.

FIG. 36 summarizes the results of these experiments which demonstratethat the suppression of gene expression observed in this in vitro assayis specific for dsRNA. These results further support the conclusion thatdsRNA suppresses gene expression in this mammalian in vitro system in amanner consistent with post-transcriptional silencing.

G. Mammalian Cells Soaked with dsRNAs Results in Gene Silencing.

Studies of post-transcriptional silencing in invertebrates havedemonstrated that transfection or injection of the dsRNA is notnecessary to achieve the suppressive affects. For example, dsRNAsuppression in C. elegans can be observed by either soaking the worms indsRNA, or by feeding the worms bacteria expressing the dsRNA ofinterest. We addressed whether dsRNA suppression in mammalian cellscould be observed without transfection of the dsRNA. Such a result wouldpresent additional potential for easily using dsRNA suppression inmammalian cells, and would also allow the use of dsRNA to suppress geneexpression in cell types which have been difficult to transfect (i.e.,cell types with a low transfection efficiency, or cell types which haveproven difficult to transfect at all).

P19 cells were grown in 6-well tissue culture plates to approximately60% confluency in growth media (αMEM/10% FBS). Varying concentrations offirefly dsRNA were added to the cultures, and cells were cultured for 12hours in growth media+dsRNA. Cells were then transfected with plasmidsexpressing firefly or sea pansy luciferase, as described in detailabove. Dual luciferase assays were carried out 12 hourspost-transfection using an Analytical Scientific Instruments model 3010Luminometer.

FIG. 37 summarizes these results which demonstrate that dsRNA cansuppress gene expression in mammalian cells without transfection.Culturing cells in the presence of dsRNA resulted in a dose dependentsuppression of firefly luciferase gene expression.

Methods:

Cell Culture. P19 mouse embryonic carcinoma cells (American Type CultureCollection, CRL-1825) were cultured in α-MEM (GIBCO/BRL) supplementedwith 10% heat-inactivated FBS and 1% antibiotic/antimycotic solution(GIBCO/BRL). Mouse embryo stem cells (J1, provided by S. Kim, ColdSpring Harbor Laboratory) were cultured in DMEM containing ESgro(Chemicon) according to the manufacturer's instructions. C2C12 murinemyoblast cells (gift of N. Tonks, Cold Spring Harbor Laboratory) werecultured in DMEM (GIBCO/BRL) supplemented with 10% heat-inactivated FBSand 1% antibiotic/antimycotic solution (GIBCO/BRL).

RNA Preparation. For the production of dsRNA, transcription templateswere generated by PCR; they contained T7 promoter sequences on each endof the template (see Hammond et al. 2000, Nature 404: 293-296). dsRNAswere prepared by using the RiboMax kit (Ambion, Austin, Tex.). Fireflyand Renilla luciferase mRNA transcripts were synthesized by using theRiboprobe kit (Promega) and were gel purified before use.

Transfection and Gene Silencing Assays. Cells were transfected withindicated amounts of dsRNA and plasmid DNA by using FuGENE6 (RocheBiochemicals) according to the manufacturer's instructions. Cells weretransfected at 50-70% confluence in 12-well plates containing either 1or 2 ml of medium per well. Dual luciferase assays (Promega) werecarried out by co-transfecting cells with plasmids contain fireflyluciferase under the control of SV40 promoter (pGL3-Control, Promega)and Renilla luciferase under the control of the SV40 earlyenhancer/promoter region (pSV40, Promega). These plasmids werecotransfected by using a 1:1 or 10:1 ratio of pGL3-control (250 ng/well)to pRL-SV40. Both ratios yielded similar results. For some experiments,cells were transfected with vectors that direct expression of enhancedgreen fluorescent protein (EGFP)-US9 fusion protein (Kalejta et al.,Exp. Cell Res. 248: 322-328, 1999) or red fluorescent protein (RFP)(pDsRed N1, CLONTECH). RNAi in S2 cells was performed as described(Hammond et al., Nature 404: 293-296, 2000).

Plasmids expressing hairpin RNAs (RNAs with a self-complimentary stemloop) were constructed by cloning the first 500 bp of the EGFP codingregion (CLONTECH) into the FLIP cassette of pRIP-FLIP as a directrepeat. The FLIP cassette contains two directional cloning sites, thesecond of which sports flanking LoxP sites (see FIG. 35A). The Zeocingene (Stratagene), present between the cloning sites, maintainsselection and, thus, stability of the FLIP cassette. The FLIP cassettecontaining EGFP direct repeats was subcloned into pcDNA3 (Invitrogen).To create an inverted repeat for hairpin production, EGFP direct repeatclones were exposed to Cre recombinase (Stratagene) in vitro and,afterward, transformed into DL759 Escherichia coli (Connelly et al.,Genes Cells 1: 285-291, 1996). These bacteria permit the replication ofDNA containing cruciform structures, which tend to form from invertedrepeats. DL759 transformants were screened for plasmids containinginverted repeats (≈50%).

Silencing of Dicer was accomplished by using a dsRNA comprising exon 25of the mouse Dicer gene and corresponding to nucleotides 5284-5552 ofthe human Dicer cDNA.

In vitro Translation and in vitro Dicer Assays. Logarithmically growingcells were harvested in PBS containing 5 mM EGTA washed twice in PBS andonce in hypotonic buffer (10 mM Hepes, pH 7.3/6 mM β-mercaptoethanol).Cells were suspended in 0.7 packed-cell volumes of hypotonic buffercontaining Complete protease inhibitors (Roche Molecular Biochemicals)and 0.5 units/ml of RNasin (Promega). Cells were disrupted in a Douncehomogenizer with a type B pestle, and lysates were centrifuged at30,000×g for 20 min. Supernatants were used in an in vitro translationassay containing capped m7G(5′)pppG firefly and Renilla luciferase mRNAor in in vitro Dicer assays containing ³²P-labeled dsRNA. For in vitrotranslation assays, 5 μl of extract were mixed with 100 ng of fireflyand Renilla mRNA along with 1 μg of dsRNA (or buffer)/10 mM DTT/0.5 mMspermidine/200 mM Hepes, 3.3 mM MgOAc/800 mM KOAc/1 mM ATP/1 mM GTP/4units of Rnasin/215 μg of creatine phosphate/1 μg of creatine phosphatekinase/1 mM amino acids (Promega). Reactions were carried out for 1 h at30° C. and quenched by adding 1× passive lysis buffer (Promega).Extracts were then assayed for luciferase activity. In vitro assays forDicer activity were performed as described (Bernstein et al., Nature409: 363-366, 2001).

Construction of Stable Silencing Lines. Ten-centimeter plates of P19cells were transfected with 5 μg of GFP hairpin expression plasmid andselected for stable integrants by using G-418 (300 ng/ml) for 14 days.Clones were selected and screened for silencing of GFP.

EXAMPLE 5 Compositions and Methods for Synthesizing siRNAs

Previous results have indicated that short synthetic RNAs (siRNAs) canefficiently induce RNA suppression. Since short RNAs do not activate thenon-specific PKR response, they offer a means for efficiently silencinggene expression in a range of cell types. However, the current state ofthe art with respect to siRNAs has several limitations. Firstly, siRNAsare currently chemically synthesized at great cost (approx. $400/siRNA).Such high costs make siRNAs impractical for either small laboratories orfor use in large scale screening efforts. Accordingly, there is a needin the art for methods for generating siRNAs at reduced cost.

We provide compositions and methods for synthesizing siRNAs by T7polymerase. This approach allows for the efficient synthesis of siRNAsat a cost consistent with standard RNA transcription reactions (approx.$16/siRNA). This greatly reduced cost makes the use of siRNA areasonable approach for small laboratories, and also will facilitatetheir use in large-scale screening projects.

FIG. 38 shows the method for producing siRNAs using T7 polymerase.Briefly, T7 polymerase is used to transcribe both a sense and antisensetranscript. The transcripts are then annealed to provide an siRNA. Oneof skill in the art will recognize that any one of the available RNApolymerases can be readily substituted for T7 to practice the invention(i.e., T3, Sp6, etc.).

This approach is amenable to the generation of a single siRNA species,as well as to the generation of a library of siRNAs. Such a library ofsiRNAs can be used in any number of high-throughput screens includingcell based phenotypic screens and gene array based screens.

EXAMPLE 6 Generation of Short Hairpin dsRNA and Suppression of GeneExpression Using such Short Hairpins

Since the realization that small, endogenously encoded hairpin RNAscould regulate gene expression via elements of the RNAi machinery, wehave sought to exploit this biological mechanism for the regulation ofdesired target genes. Here we show that short hairpin RNAs (shRNAs) caninduce sequence-specific gene silencing in mammalian cells. As isnormally done with siRNAs, silencing can be provoked by transfectingexogenously synthesized hairpins into cells. However, silencing can alsobe triggered by endogenous expression of shRNAs. This observation opensthe door to the production of continuous cells lines in which RNAi isused to stably suppress gene expression in mammalian cells. Furthermore,similar approaches should prove efficacious in the creation oftransgenic animals and potentially in therapeutic strategies in whichlong-term suppression of gene function is essential to produce a desiredeffect.

Several groups (Grishok et al., Cell 106: 23-34, 2001; Ketting et al.,Genes & Dev. 15: 2654-2659, 2001; Knight et al., Science 293: 2269-2271,2001; Hutvagner et al., Science 293: 834-838, 2001) have shown thatendogenous triggers of gene silencing, specifically small temporal RNAs(stRNAs) let-7 and lin-4, function at least in part through RNAipathways. Specifically, these small RNAs are encoded by hairpinprecursors that are processed by Dicer into mature, ˜21-nt forms.Moreover, genetic studies in C. elegans have shown a requirement forArgonaute-family proteins in stRNA function. Specifically, alg-1 andalg-2, members of the EIF2c subfamily, are implicated both in stRNAprocessing and in their downstream effector functions (Grishok et al.,2001, supra). We have recently shown that a component of RISC, theeffector nuclease of RNAi, is a member of the Argonaute family,prompting a model in which stRNAs may function through RISC-likecomplexes, which regulate mRNA translation rather than mRNA stability(Hammond et al., Science 293: 1146-1150, 2001).

A. Short Hairpin RNAs Triggeedr Gene Silencing in Drosophila Cells.

We wished to test the possibility that we might retarget these small,endogenously encoded hairpin RNAs to regulate genes of choice with theultimate goal of subverting this regulatory system for manipulating geneexpression stably in mammalian cell lines and in transgenic animals.Whether triggered by long dsRNAs or by siRNAs, RNAi is generally morepotent in the suppression of gene expression in Drosophila S2 cells thanin mammalian cells. We therefore chose this model system in which totest the efficacy of short hairpin RNAs (shRNAs) as inducers of genesilencing.

Neither stRNAs nor the broader group of miRNAs that has recently beendiscovered form perfect hairpin structures. Indeed, each of these RNAsis predicted to contain several bulged nucleotides within their rathershort (˜30-nt) stem structures. Because the position and character ofthese bulged nucleotides have been conserved throughout evolution andamong at least a subset of miRNAs, we sought to design retargeted miRNAmimics to conserve these predicted structural features. Only the let-7and lin-4 miRNAs have known mRNA targets (Wightman et al., Cell 75:855-862, 1993; Slack et al., Mol. Cell 5: 659-669, 2000). In both cases,pairing to binding sites within the regulated transcripts is imperfect,and in the case of lin-4, the presence of a bulged nucleotide iscritical to suppression (Ha et al., Genes & Dev. 10: 3041-3050, 1996).We therefore also designed shRNAs that paired imperfectly with theirtarget substrates. A subset of these shRNAs is depicted in FIG. 39A.

To permit rapid testing of large numbers of shRNA variants andquantitative comparison of the efficacy of suppression, we chose to usea dual-luciferase reporter system, as previously described for assays ofRNAi in both Drosophila extracts (Tuschl et al., Genes & Dev. 13:3191-3197, 1999) and mammalian cells (Caplen et al., Proc. Natl. Acad.Sci. 98: 9742-9747, 2001; Elbashir et al., Nature 411: 494-498, 2001).Cotransfection of firefly and Renilla luciferase reporter plasmids witheither long dsRNAs or with siRNAs homologous to the firefly luciferasegene yielded an ˜95% suppression of firefly luciferase without effect onRenilla luciferase (FIG. 39B; data not shown). Firefly luciferase couldalso be specifically silenced by co-transfection with homologous shRNAs.The most potent inhibitors were those composed of simple hairpinstructures with complete homology to the substrate. Introduction of G-Ubasepairs either within the stem or within the substrate recognitionsequence had little or no effect (FIG. 39A and 39B; data not shown).

These results show that short hairpin RNAs can induce gene silencing inDrosophila S2 cells with potency similar to that of siRNAs (FIG. 39B).However, in our initial observation of RNA interference in Drosophila S2cells, we noted a profound dependence of the efficiency of silencing onthe length of the dsRNA trigger (Hammond et al., Nature 404: 293-296,2000). Indeed, dsRNAs of fewer than ˜200 nt triggered silencing veryinefficiently. Silencing is initiated by an RNase III family nuclease,Dicer, that processes long dsRNAs into ˜22-nt siRNAs. In accord withtheir varying potency as initiators of silencing, long dsRNAs areprocessed much more readily than short RNAs by the Dicer enzyme(Bernstein et al., Nature 409: 363-366, 2001). We therefore testedwhether shRNAs were substrates for the Dicer enzyme.

We had noted previously that let-7 (Ketting et al., Genes & Dev. 15:2654-2659, 2001) and other miRNAs (E. Bernstein, unpublished data) areprocessed by Dicer with an unexpectedly high efficiency as compared withshort, nonhairpin dsRNAs. Similarly, Dicer efficiently processed shRNAsthat targeted firefly luciferase, irrespective of whether they weredesigned to mimic a natural Dicer substrate (let-7) or whether they weresimple hairpin structures (FIG. 39C). These data suggest thatrecombinant shRNAs can be processed by Dicer into siRNAs and areconsistent with the idea that these short hairpins trigger genesilencing via an RNAi pathway.

B. Short Hairpin RNAs Activated Gene Silencing in Mammalian Cells.

Mammalian cells contain several endogenous systems that were predictedto hamper the application of RNAi. Chief among these is adsRNA-activated protein kinase, PKR, which effects a general suppressionof translation via phosphorylation of EIF-2α (Williams, Biochem. Soc.Trans. 25: 509-513, 1997; Gil et al., Apoptosis 5: 107-114, 2000).Activation of these, and other dsRNA-responsive pathways, generallyrequires duplexes exceeding 30 bp in length, possibly to permitdimerization of the enzyme on its allosteric activator (e.g., Clarke etal., RNA 1: 7-20, 1995). Small RNAs that mimic Dicer products, siRNAs,presumably escape this limit and trigger specific silencing, in partbecause of their size. However, short duplex RNAs that lack signaturefeatures of siRNAs can efficiently induce silencing in Drosophila S2cells but not in mammalian cells (A. A. Caudy, unpublished data).Endogenously encoded miRNAs may also escape PKR surveillance because oftheir size but perhaps also because of the discontinuity of their duplexstructure. Given that shRNAs of <30 bp were effective inducers of RNAiin Drosophila S2 cells, we tested whether these RNAs could also inducesequence-specific silencing in mammalian cells.

Human embryonic kidney (HEK293T) cells were cotransfected withchemically synthesized shRNAs and with a mixture of firefly and Renillaluciferase reporter plasmids. As had been observed in S2 cells, shRNAswere effective inducers of gene silencing. Once again, hairpins designedto mimic let-7 were consistently less effective than were simple hairpinRNAs, and the introduction of mismatches between the antisense strand ofthe shRNA and the mRNA target abolished silencing (FIG. 40A; data notshown). Overall, shRNAs were somewhat less potent silencing triggersthan were siRNAs. Whereas siRNAs homologous to firefly luciferaseroutinely yielded ˜90%-95% suppression of gene expression, suppressionlevels achieved with shRNAs ranged from 80%-90% on average. As we alsoobserve with siRNAs, the most important determinant of the potency ofthe silencing trigger is its sequence. We find that roughly 50% of bothsiRNAs and shRNAs are competent for suppressing gene expression.However, neither analysis of the predicted structures of the target mRNAnor analysis of alternative structures in siRNA duplexes or shRNAhairpins has proved of predictive value for choosing effectiveinhibitors of gene expression.

We have adopted as a standard, shRNA duplexes containing 29 bp. However,the size of the helix can be reduced to ˜25 nt without significant lossof potency. Duplexes as short as 22 bp can still provoke detectablesilencing, but do so less efficiently than do longer duplexes. In nocase did we observe a reduction in the internal control reporter(Renilla luciferase) that would be consistent with an induction ofnonspecific dsRNA responses.

The ability of shRNAs to induce gene silencing was not confined to 293Tcells. Similar results were also obtained in a variety of othermammalian cell lines, including human cancer cells (HeLa), transformedmonkey epithelial cells (COS-1), murine fibroblasts (NIH 3T3), anddiploid human fibroblasts (IMR90; FIG. 40; data not shown).

C. Synthesis of Effective Inhibitors of Gene Expression using T7 RNAPolymerse.

The use of siRNAs to provoke gene silencing is developing into astandard methodology for investigating gene function in mammalian cells.To date, siRNAs have been produced exclusively by chemical synthesis(e.g., Caplen et al., Proc. Natl. Acad. Sci. 98: 9742-9747, 2001;Elbashir et al., Nature 411: 494-498, 2001). However, the costsassociated with this approach are significant, limiting its potentialutility as a tool for investigating in parallel the functions of largenumbers of genes. Short hairpin RNAs are presumably processed intoactive siRNAs in vitro by Dicer. Thus, these may be more tolerant ofterminal structures, both with respect to nucleotide overhangs and withrespect to phosphate termini. We therefore tested whether shRNAs couldbe prepared by in vitro transcription with T7 RNA polymerase.

Transcription templates that were predicted to generate siRNAs andshRNAs similar to those prepared by chemical RNA synthesis were preparedby DNA synthesis (FIG. 41A,C). These were tested for efficacy both in S2cells (data not shown) and in human 293 cells (FIG. 41B,D). Overall, theperformance of the T7-synthesized hairpin or siRNAs closely matched theperformance of either produced by chemical synthesis, both with respectto the magnitude of inhibition and with respect to the relativeefficiency of differing sequences. Because T7 polymerase prefers toinitiate at twin guanosine residues, however, it was critical toconsider initiation context when designing in vitro transcribed siRNAs(FIG. 41B). In contrast, shRNAs, which are processed by Dicer (see FIG.39C), tolerate the addition of these bases at the 5′ end of thetranscript.

Studies in Drosophila embryo extracts indicate that siRNAs possess 5′phosphorylated termini, consistent with their production by an RNase IIIfamily nuclease. In vitro, this terminus is critical to the induction ofRNAi by synthetic RNA oligonucleotides (Elbashir et al., EMBO J. 20:6877-6888, 2001; Nykanen et al., Cell 107: 309-321, 2001). Chemicallysynthesized siRNAs are nonphosphorylated, and enzymatic addition of a 5′phosphate group in vitro prior to transfection does not increase thepotency of the silencing effect (A. A. Caudy, unpublished data). Thissuggests either that the requirement for phosphorylated termini is lessstringent in mammalian cells or that a kinase efficiently phosphorylatessiRNAs in vitro. RNAs synthesized with T7 RNA polymerase, however,possess 5′ triphosphate termini. We therefore explored the possibilityof synthesizing siRNAs with T7 polymerase followed by treatment in vitrowith pyrophosphatase to modify the termini to resemble those of siRNAs.Surprisingly, monophosphorylated siRNAs (data not shown) were as potentin inducing gene silencing as transcription products bearingtriphosphate termini (FIG. 41B). This may suggest either that therequirement for monophosphorylated termini is less stringent inmammalian cells or that siRNAs are modified in vitro to achieve anappropriate terminal structure.

Considered together, our data suggest that both shRNAs and siRNAduplexes can be prepared by synthesis with T7 RNA polymerase in vitro.This significantly reduces the cost of RNAi in mammalian cells and pavesthe way for application of RNAi on a whole-genome scale.

D. Transcription of Small Hairpin RNAs in vitro by RNA Polymerase III.

Although siRNAs are an undeniably effective tool for probing genefunction in mammalian cells, their suppressive effects are by definitionof limited duration. Delivery of siRNAs can be accomplished by any of anumber of transient transfection methodologies, and both the timing ofpeak suppression and the recovery of protein levels as silencing decayscan vary with both the cell type and the target gene. Therefore, onelimitation on siRNAs is the development of continuous cell lines inwhich the expression of a desired target is stably silenced.

Hairpin RNAs, consisting of long duplex structures, have been proved aseffective triggers of stable gene silencing in plants, in C. elegans,and in Drosophila (Kennerdell et al., Nat. Biotechnol. 18: 896-898,2000; Smith et al., Nature 407: 319-320, 2000; Tavernarakis et al., Nat.Genet. 24: 180-183, 2000). We have recently shown stable suppression ofgene expression in cultured mammalian cells by continuous expression ofa long hairpin RNA (Paddison et al., Proc. Natl. Acad. Sci. 99:1443-1448, 2002). However, the scope of this approach was limited by thenecessity of expressing such hairpins only in cells that lack adetectable PKR response. In principle, shRNAs could bypass suchlimitations and provide a tool for evoking stable suppression by RNA inmammalian somatic cells.

To test this possibility, we initially cloned sequences encoding afirefly luciferase shRNA into a CMV-based expression plasmid. This waspredicted to generate a capped, polyadenylated RNA polymerase IItranscript in which the hairpin was extended on both the 5′ and 3′ endsby vector sequences and poly(A). This construct was completely inert insilencing assays in 293T cells.

During our studies on chemically and T7-synthesized shRNAs, we notedthat the presence of significant single-stranded extensions (either 5′or 3′ of the duplex) reduced the efficacy of shRNAs. We thereforeexplored the use of alternative promoter strategies in an effort toproduce more defined hairpin RNAs. In particular, RNA polymerase IIIpromoters have well-defined initiation and termination sites andnaturally produce a variety of small, stable RNA species. Although manyPol III promoters contain essential elements within the transcribedregion, limiting their utility for our purposes; class III promoters useexclusively nontranscribed promoter sequences. Of these, the U6 snRNApromoter and the H1 RNA promoter have been well studied (Lobo et al.,Nucleic Acids Res. 18: 2891-2899, 1990; Hannon et al., J. Biol. Chem.266: 22796-22799, 1991; Chong et al., J. Biol. Chem. 276: 20727-20734,2001).

By placing a convenient cloning site immediately behind the U6 snRNApromoter, we have constructed pShh-1, an expression vector in whichshort hairpins are harnessed for gene silencing. Into this vector eitherof two shRNA sequences derived from firefly luciferase were cloned fromsynthetic oligonucleotides. These were cotransfected with firefly andRenilla luciferase expression plasmids into 293T cells. One of the twoencoded shRNAs provoked effective silencing of firefly luciferasewithout altering the expression of the internal control (FIG. 42C). Thesecond encoded shRNA also produced detectable, albeit weak, repression.In both cases, silencing was dependent on insertion of the shRNA in thecorrect orientation with respect to the promoter (FIG. 42C; data notshown). Although the shRNA itself is bilaterally symmetric, insertion inthe incorrect orientation would affect Pol III termination and ispredicted to produce a hairpin with both 5′ and 3′ single-strandedextensions. Similar results were also obtained in a number of othermammalian cell lines including HeLa, COS-1, NIH 3T3, and IMR90 (FIG. 42;data not shown). pShh1-Ff1 was, however, incapable of effectingsuppression of the luciferase reporter in Drosophila cells, in which thehuman U6 promoter is inactive.

E. Dicer Is Required for shRNA-Mediated Gene Silencing.

As a definitive test of whether the plasmid-encoded shRNAs brought aboutgene silencing via the mammalian RNAi pathway, we assessed thedependence of suppression on an essential component of the RNAi pathway.We transfected pShh1-Ff1 along with an siRNA homologous to human Dicer.FIG. 43 shows that treatment of cells with Dicer siRNAs is able tocompletely depress the silencing induced by pShh1-Ff1. Addition of anunrelated siRNA had no effect on the magnitude of suppression bypShh1-Ff1. Importantly, Dicer siRNAs had no effect on siRNA-inducedsilencing of firefly luciferase. These results are consistent withshRNAs operating via an RNAi pathway similar to those provoked by stRNAsand long dsRNAs. Furthermore, it suggests that siRNA-mediated silencingis less sensitive to depletion of the Dicer enzyme.

F. Stable shRNA-Mediated Gene Silencing of an Endogenous Gene.

The ultimate utility of encoded short hairpins will be in the creationof stable mutants that permit the study of the resulting phenotypes. Wetherefore tested whether we could create a cellular phenotype throughstable suppression. Expression of activated alleles of the ras oncogenein primary mouse embryo fibroblasts (MEFs) induces a stable growtharrest that resembles, as a terminal phenotype, replicative senescence(Serrano et al., Cell 88: 593-602, 1997). Cells cease dividing andassume a typical large, flattened morphology. Senescence can becountered by mutations that inactivate the p53 tumor suppressor pathway(Serrano et al. 1997, supra). As a test of the ability of vector-encodedshRNAs to stably suppress an endogenous cellular gene, we generated ahairpin that was targeted to the mouse p53 gene. As shown in FIG. 44,MEFs transfected with pBabe-RasV12 fail to proliferate and show asenescent morphology when cotransfected with an empty control vector. Asnoted previously by Serrano et al., the terminally arrested state isachieved in 100% of drug-selected cells in culture by 8 dpost-transfection. However, upon cotransfection of an activated rasexpression construct with the pShh-p53, cells emerged from drugselection that not only fail to adopt a senescent morphology but alsomaintain the ability to proliferate for a minimum of several weeks inculture (FIG. 44). These data strongly suggest that shRNA expressionconstructs can be used for the creation of continuous mammalian celllines in which selected target genes are stably suppressed.

G. Simultaneous Introduction of Multiple Hairpin RNAs does not ProduceSynergy.

In an attempt to further understand the mechanisms by which shorthairpins suppress gene expression, we examined the effects oftransfecting cells with a mixture of two different short hairpinscorresponding to firefly luciferase. FIG. 45 summarizes the results ofexperiments which suggest that there is no synergistic affects onsuppression of firefly luciferase gene expression obtained when cellsare exposed to a mixture of such short hairpins.

Methods:

Cell culture. HEK 293T, HeLa, COS-1, MEF, and IMR90 cells were culturedin DMEM (GIBCO BRL) supplemented with 10% heat-inactivated fetal bovineserum (FBS) and 1% antibiotic/antimycotic solution (GIBCO BRL). NIH 3T3cells were cultured in DMEM supplemented with 10% heat-inactivated calfserum and 1% antibiotic/antimycotic solution.

RNA preparation. Both shRNAs and siRNAs were produced in vitro usingchemically synthesized DNA oligonucleotide templates (Sigma) and the T7Megashortscript kit (Ambion). Transcription templates were designed suchthat they contained T7 promoter sequences at the 5′ end. shRNAtranscripts subjected to in vitro Dicer processing were synthesizedusing a Riboprobe kit (Promega). Chemically synthesized RNAs wereobtained from Dharmacon, Inc.

Transfection and gene silencing assays. Cells were transfected withindicated amounts of siRNA, shRNA, and plasmid DNA using standardcalcium phosphate procedures at 50%-70% confluence in 6-well plates.Dual luciferase assays (Promega) were carried out by cotransfectingcells with plasmids containing firefly luciferase under the control ofthe SV40 promoter (pGL3-Control, Promega) and Renilla luciferase underthe control of the SV40 early enhancer/promoter region (pSV40, Promega).Plasmids were cotransfected using a 1:1 ratio of pGL3-Control (250ng/well) to pRL-SV40. RNAi in S2 cells was performed as previouslydescribed (Hammond et al., Nature 404: 293-296, 2000). For stablesilencing, primary MEFs (a gift from S. Lowe, Cold Spring HarborLaboratory, N.Y.) were cotransfected using Fugene 6 with pBabe-Ha-rasV12and pShh-p53 (no resistance marker), according to the manufacturer'srecommendations. Selection was for the presence of the activatedHa-rasV12 plasmid, which carries a puromycin-resistance marker. ThepShh-p53 plasmid was present in excess, as is standard in acotransfection experiment. We have now generated a version of the U6promoter vector (pSHAG-1) that is compatible with the GATEWAY system(Invitrogen), and this can be used to transport the shRNA expressioncassette into a variety of recipient vectors that carry cis-linkedselectable markers. Furthermore, we have validated delivery of shRNAsusing retroviral vectors. Updated plasmid information can be obtainedat:

http://www.cshl.org/public/science/hannon.html.

Plasmids expressing hairpin RNAs. The U6 promoter region from −265 to +1was amplified by PCR, adding 5′ KpnI and 3′ EcoRV sites for cloning intopBSSK⁺. A linker/terminator oligonucleotide set bearing the U6terminator sequence and linker ends of 5′ EcoRV and 3′ NotI was clonedinto the promoter construct, resulting in a U6 cassette with an EcoRVsite for insertion of new sequences. This vector has been named pShh1.Blunt-ended, double-stranded DNA oligonucleotides encoding shRNAs withbetween 19 and 29 bases of homology to the targeted gene were ligatedinto the EcoRV site to produce expression constructs. Theoligonucleotide sequence used to construct Ff1 was:TCCAATTCAGCGGGAGCCACCTGATGAAGCTTGATCGGGTGGCTCTCGCTGAGTT GGAATCCATTTTTTTT(SEQ ID NO: 38). This sequence is preceded by the sequence GGAT, whichis supplied by the vector, and contains a tract of more than five Ts asa Pol III terminator.

In vitro Dicer assays. In vitro assays for Dicer activity were performedas described (Bernstein et al., Nature 409: 363-366, 2001).

EXAMPLE 7 Encoded Short Hairpins Function in vitro

An object of the present invention is to improve methods for generatingsiRNAs and short hairpins for use in specifically suppressing geneexpression. Example 6 demonstrates that siRNAs and short hairpins arehighly effective in specifically suppressing gene expression.Accordingly, it would be advantageous to combine the efficientsuppression of gene expression attainable using short hairpins andsiRNAs with a method to encode such RNA on a plasmid and express iteither transiently or stably.

FIG. 46 demonstrates that short hairpins encoded on a plasmid areeffective in suppressing gene expression. DNA oligonucleotides encoding29 nucleotide hairpins corresponding to firefly luciferase were insertedinto a vector containing the U6 promoter. Three independent constructswere examined for their ability to specifically suppress fireflyluciferase gene expression in 293T cells. siOligo1-2, siOligo1-6, andsiOligo1-19 (construct in the correct orientation) each suppressed geneexpression as effectively as siRNA. In contrast, siOligo1-10 (constructin the incorrect orientation) did not suppress gene expression.Additionally, an independent construct targeted to a different portionof the firefly luciferase gene did not effectively suppress geneexpression in either orientation (siOligo2-23, siOligo2-36).

The results summarized in FIG. 46 demonstrate that transient expressionof siRNAs and short hairpins encoded on a plasmid can efficientlysuppress gene expression. One of skill can choose from amongst a rangeof vectors to either transiently or stably express an siRNA or shorthairpin. Non-limiting examples of vectors and strategies to stablyexpress short dsRNAs are presented in FIGS. 47-49.

EXAMPLE 8 dsRNA Suppression in the Absence of a PKR Response

One potential impediment to the use of RNAi to suppress gene expressionin some cell types, is the non-specific PKR response that can betriggered by long dsRNAs. Numerous mammalian viruses have evolved theability to block PKR in order to aid in the infection of potential hostcells. For example, adenoviruses express RNAs which mimic dsRNA but donot activate the PKR response. Vaccinia virus uses two strategies toevade PKR: the expression of E3L which binds and masks dsRNA; theexpression of K3L to mimic the natural PKR substrate eIF2α.

Our understanding of the mechanisms by which viruses avoid the PKRresponse allows us to design approaches to circumvent the PKR responsein cell types in which in might be advantageous to suppression geneexpression with long dsRNAs. Possible approaches include treating cellswith an agent that inhibits protein kinase RNA-activated (PKR)apoptosis, such as by treatment with agents which inhibit expression ofPKR, cause its destruction, and/or inhibit the kinase activity of PKR.Accordingly, RNAi suppression of gene expression in such cell typescould involve first inhibiting the PKR response, and then delivering adsRNA identical or similar to a target gene.

A. In a murine myoblast cell line, C2C12, we noted that the cellsresponded to long dsRNAs with a mixture of specific and non-specific(presumably PKR) responses. In order to attenuate the non-specific PKRresponse while maintaining the robust and specific suppression due tothe long dsRNA, C2C12 cells were transfected with a vector that directsK3L expression. This additional step successfully attenuated the PKRresponse, however expression of K3L protein had no effect on themagnitude of specific inhibition.

B. However, since the efficacy of such a two step approach had not beenpreviously demonstrated, it was formerly possible that dsRNA suppressionwould not be possible in cells with a PKR response. FIG. 50 summarizesresults which demonstrate that such a two step approach is possible, andthat robust and specific dsRNA mediated suppression is possible in cellswhich had formerly possessed a robust PKR response.

Briefly, dual luciferase assay were carried out as described in detailabove. The experiments were carried out using PKR^(−/−) MEFs harvestedfrom E13.5 PKR^(−/−) mouse embryos. MEFs typically have a robust PKRresponse, and thus treatment with long dsRNAs typically results innon-specific suppression of gene expression and apoptosis. However, inPKR^(−/−) cells examined 12, 42, and 82 hours after transfection,expression of dsRenilla luciferase RNA specifically suppressesexpression Renilla reniformis (sea pansy) luciferase. This suppressionis stable over time.

These results demonstrate that the non-specific PKR response can beblocked without affecting specific suppression of gene expressionmediated by dsRNA. This allows the use of long dsRNAs to suppress geneexpression in a diverse range of cell types, including those that wouldbe previously intractable due to the confounding influences of thenon-specific PKR response to long dsRNA.

EXAMPLE 9 Suppression of Gene Expression using dsRNA which Correspondsto Non-Coding Sequence

Current models for the mechanisms which drive RNAi have suggested thatthe dsRNA construct must contain coding sequence corresponding to thegene of interest. Although evidence has demonstrated that such codingsequence need not be a perfect match to the endogenous coding sequence(i.e., it may be similar), it has been widely held that the dsRNAconstruct must correspond to coding sequence. We present evidence thatcontradicts the teachings of the prior art, and demonstrate that dsRNAcorresponding to non-coding regions of a gene can suppress gene functionin vitro. These results are significant not only because theydemonstrate that dsRNA identical or similar to non-coding sequences(i.e., promoter sequences, enhancer sequences, or intronic sequences)can mediate suppression, but also because we demonstrate the in vitrosuppression of gene expression using dsRNA technology in a mouse model.

We generated doubled stranded RNA corresponding to four segments of themouse tyrosinase gene promoter. Three of these segments correspond tothe proximal promoter and one corresponds to an enhancer (FIG. 51). Thetyrosinase gene encodes the rate limiting enzyme involved in the melaninbiosynthetic pathway (Bilodeau et al., Pigment Cell Research 14:328-336, 2001). Accordingly, suppression of the tyrosinase gene isexpected to inhibit pigmentation.

Double stranded RNA corresponding to each of the above promoter segmentswas injected into the pronuclei of fertilized eggs. Pups were born after19 days. In total 42/136 (31%) of the embryos were carried to term. Thisnumber is within the expected range for transgenesis (30-40%). Two pupsout of 42 (5%) appear totally unpigmented at birth, consistent withsuppression of tyrosinase function.

Methods:

dsRNA from non-coding promoter region of tyrosinase gene. Four segmentsof the mouse tyrosinase gene promoter were amplified by PCR usingprimers which incorporated T7 RNA polymerase promoters into the PCRproducts (shown in bold—FIG. 51). Sequences of the mouse tyrosinase gene5′ flanking regions were obtained from GenBank (accession number D00439and X51743). The sequence of the tyrosinase enhancer, locatedapproximately 12 kb upstream of the transcriptional start site, was alsoobtained from GenBank (accession number X76647).

The sequences of the primers used were as follows: note the sequence ofthe T7 RNA polymerase promoter is shown in bold: (a) Tyrosinase enhancer(˜12 kb upstream): (SEQ ID NO: 39)5′ TAATACGACTCACTATAGGGCAAGGTCATAGTTCCTGCCAGCTG 3′ (SEQ ID NO: 40)5′ TAATACGACTCACTATAGGGCAGATATTTTCTTACCACCCACCC 3′ (b) −1404 to −1007:(SEQ ID NO: 41) 5′ TAATACGACTCACTATAGGGTTAAGTTTAACAGGAGAAGCTGGA 3′ (SEQID NO: 42) 5′ TAATACGACTCACTATAGGGAAATCATTGCTTTCCTGATAATGC 3′ (c) −1003to −506: (SEQ ID NO: 43) 5′ TAATACGACTCACTATAGGGTAGATTTCCGCAGCCCCAGTGTTC3′ (SEQ ID NO: 44) 5′ TAATACGACTCACTATAGGGGTTGCCTCTCATTTTTCCTTGATT 3′(d) −505 to −85: (SEQ ID NO: 45)5′ TAATACGACTCACTATAGGGTATTTTAGACTGATTACTTTTATAA 3′ (SEQ ID NO: 46)5′ TAATACGACTCACTATAGGGTCACATGTTTTGGCTAAGACCTAT 3′

PCR products were gel purified from 1% TAE agarose gels using QiaExIIGel Extraction Kit (Qiagen). Double stranded RNA was produced from thesetemplates using T7-Megashortscript Kit (Ambion). Enzymes andunincorporated nucleotides were removed using Qiaquick MinElute PCRPurification Kit. RNA was phenol/chloroform extracted twice, and ethanolprecipitated. Pellets were resuspended in injection buffer ((10 mM Tris(pH 7.5), 0.15 nM EDTA (pH 8.0)) at a concentration of 20 ng/ul and runon a 1% TAE agarose gel to confirm integrity.

Generation of mice: An equal mixture of double stranded RNA from each ofthe above primer sets was injected into the pronuclei of fertilized eggsfrom C57BL6J mice. A total of 136 injections was performed, and 34embryos were implanted into each of 4 pseudopregnant CD-1 females. Pupswere born after 19 days. In total, 42/136 (31%) of the embryos werecarried to term. 2/42 pups (5%) appear totally unpigmented at birth.

It is not clear whether the RNAi mediated by dsRNA identical or similarto non-coding sequence works via the same mechanism as PTGS observed inthe presence of dsRNA identical or similar to coding sequence. However,whether these results ultimately reveal similar or differing mechanismsdoes not diminish the tremendous utility of the compositions and methodsof the present invention to suppress expression of one or more genes invitro or in vitro.

The present invention demonstrates that dsRNA ranging in length from20-500 nt can readily suppress expression of target genes both in vitroand in vitro. Furthermore, the present invention demonstrates that thedsRNAs can be generated using a variety of methods including theformation of hairpins, and that these dsRNAs can be expressed eitherstably or transiently. Finally, the present invention demonstrates thatdsRNA identical or similar to non-coding sequences can suppress targetgene expression.

EXAMPLE 10 RNA Interference in Adult Mice

RNA interference is an evolutionarily conserved surveillance mechanismthat responds to double-stranded RNA by sequence-specific silencing ofhomologous genes. Here we show that transgene expression can besuppressed in adult mice by synthetic small interfering RNAs and bysmall-hairpin RNAs transcribed in vitro from DNA templates. We also showthe therapeutic potential of this technique by demonstrating effectivetargeting of a sequence from hepatitis C virus by RNA interference invitro.

Small interfering RNAs (siRNAs) mimic intermediates in theRNA-interference (RNAi) pathway and can silence genes in somatic cellswithout activating non-specific suppression by double-strandedRNA-dependent protein kinase (Elbashir et al., Nature 411: 494-498,2001). To investigate whether siRNAs also inhibit gene expression invitro, we used a modification of hydrodynamic transfection methods(Zhang et al., Hum. Gene Therapy 10: 1735-1737, 1999; Liu et al., GeneTherapy 6: 1258-1266, 1999; Chang et al., J. Virol. 75: 3469-3473, 2001)to deliver naked siRNAs to the livers of adult mice. Either an siRNAderived from firefly luciferase or an unrelated siRNA was co-injectedwith a luciferase-expression plasmid (for construct description andsequences, see FIG. 52). We monitored luciferase expression in livinganimals using quantitative whole-body imaging (Contag, et al.,Photochem. Photobiol. 66: 523-531, 1997) (see FIG. 53 a, 54 a), andfound that it was dependent on reporter-plasmid dose.

In each experiment, serum measurements of a co-injected human α-1antitrypsin (hAAT) plasmid (Yant et al., Nature Genet. 25: 35-41, 2000)served to normalize transfection efficiency and to monitor non-specifictranslational inhibition. Average serum concentrations of hAAT after 74h were similar in all groups.

Our results indicate that there was specific, siRNA-mediated inhibitionof luciferase expression in adult mice (P<0.0115) and that unrelatedsiRNAs had no effect (P<0.864; FIG. 53 a, 53 b). In 11 independentexperiments, luciferase siRNAs reduced luciferase expression (as judgedby emitted light) by an average of 81% (±2.2%). These findings indicatethat RNAi can downregulate gene expression in adult mice.

As RNAi degrades respiratory syncitial virus RNAs in culture (Bitko etal. 2001, BMC Microbiol. 1: 34), we investigated whether RNAi could bedirected against a human pathogenic RNA expressed in a mouse, namelythat of hepatitis C virus (HCV). Infection by HCV (an RNA virus thatinfects 1 in 40 people worldwide) is the most common reason for livertransplantation in the United States and Europe. We fused the NS5Bregion (non-structural protein 5B, viral-polymerase-encoding region) ofthis virus with luciferase RNA and monitored RNAi by co-transfection invitro. An siRNA targeting the NS5B region reduced luciferase expressionfrom the chimaeric HCV NS5B protein-luciferase fusion by 75% (±6.8%; 6animals per group). This result suggests that it may be feasible to useRNAi as a therapy against other important human pathogens.

Although our results show that siRNAs are functional in mice, deliveryremains a major obstacle. Unlike siRNAs, functional small-hairpin RNAs(shRNAs) can be expressed in vitro from DNA templates using RNApolymerase III promoters (Paddison et al., Genes Dev. 16: 948-958, 2002;Tuschl, Nature Biotechnol. 20: 446-448, 2002); they are as effective assiRNAs in inducing gene suppression. Expression of a cognate shRNA(pShh1-Ff1) inhibited luciferase expression by up to 98% (±0.6%), withan average suppression of 92.8% (±3.39%) in three independentexperiments (see FIG. 54 a, 54 b). An empty shRNA-expression vector hadno effect; reversing the orientation of the shRNA (pShh1-Ff1rev) insertprevents gene silencing because it alters the termination by RNApolymerase III and generates an improperly structured shRNA. Thesefindings indicate that plasmid-encoded shRNAs can induce a potent andspecific RNAi response in adult mice.

RNAi may find application in functional genomics or in identifyingtargets for designer drugs. It is a more promising system thangene-knockout mice because groups of genes can be simultaneouslyrendered ineffective without the need for time-consuming crosses. Genetherapy currently depends on the ectopic expression of exogenousproteins; however, RNAi may eventually complement this gain-of-functionapproach by silencing disease-related genes with DNA constructs thatdirect the expression of shRNAs. Our method of RNAi delivery could alsobe tailored to take advantage of developing viral and non-viralgene-transfer vectors in a clinical context.

EXAMPLE 11 Germ-Line Transmission of RNAi in Mice

MicroRNA molecules (miRNAs) are small, noncoding RNA molecules that havebeen found in a diverse array of eukaryotes, including mammals. miRNAprecursors share a characteristic secondary structure, forming short‘hairpin’ RNAs. Genetic and biochemical studies have indicated thatmiRNAs are processed to their mature forms by Dicer, an RNAse III familynuclease, and function through RNA-mediated interference (RNAi) andrelated pathways to regulate the expression of target genes (Hannon,Nature 418: 244-251, 2002; Pasquinelli et al., Annu. Rev. Cell. Dev.Biol. 18: 495-513, 2002). Recently, we and others have remodeled miRNAsto permit experimental manipulation of gene expression in mammaliancells and have dubbed these synthetic silencing triggers ‘short hairpinRNAs’ (shRNAs) (Paddison et al., Cancer Cell 2: 17-23, 2002). Silencingby shRNAs requires the RNAi machinery and correlates with the productionof small interfering RNAs (siRNAs), which are a signature of RNAi.

Expression of shRNAs can elicit either transient or stable silencing,depending upon whether the expression cassette is integrated into thegenome of the recipient cultured cell (Paddison et al., Cancer Cell 2:17-23, 2002). shRNA expression vectors also induce gene silencing inadult mice following transient delivery (Lewis et al., Nat. Genet. 32:107-108, 2002; McCaffrey et al., Nature 418: 38-39, 2002). However, forshRNAs to be a viable genetic tool in mice, stable manipulation of geneexpression is essential. Hemann and colleagues have demonstratedlong-term suppression of gene expression in vitro following retroviraldelivery of shRNA-expression cassettes to hematopoietic stem cells(Hemann et al., Nat. Genet. in the press, 2003). Here we sought to testwhether shRNA-expression cassettes that were passed through the mousegerm-line could enforce heritable gene silencing.

We began by taking standard transgenesis approaches (Gordon et al.,Methods Enzymol. 225: 747-771, 1993) using shRNAs directed against avariety of targets with expected phenotypes, including the genesencoding tyrosinase (albino), myosin VIIa (shaker), Bmp-5 (crinkledears), Hox a-10 (limb defects), homogentisate 1,2,-dioxygenase.(urineturns black upon exposure to air), Hairless (hair loss) and melanocortin1 receptor (yellow). Three constructs per gene were linearized andinjected into pronuclei to produce transgenic founder animals. Althoughwe noted the presence of the transgene in some animals, virtually noneshowed a distinct or reproducible phenotype that was expected for ahypomorphic allele of the targeted gene.

Therefore, we decided to take another approach: verifying the presenceof the shRNA and its activity toward a target gene in cultured embryonicstem (ES) cells and then asking whether those cells retained suppressionin a chimeric animal in vitro. We also planned to test whether suchcells could pass a functional RNAi-inducing construct through the mousegerm-line. For these studies, we chose to examine a novel gene, Neil1,which is proposed to have a role in DNA repair. Oxidative damageaccounts for 10,000 DNA lesions per cell per day in humans and isthought to contribute to carcinogenesis, aging and tissue damagefollowing ischemia (Ames et al., Proc. Natl. Acad. Sci. USA 90:7915-7922, 1993; Jackson et al., Mutat. Res. 477: 7-21, 2001). OxidativeDNA damage includes abasic sites, strand breaks and at least 20 oxidizedbases, many of which are cytotoxic or pro-mutagenic (Dizdaroglu et al.,Free Radic. Biol. Med. 32: 1102-1115, 2002). DNA N-glycosylases initiatethe base excision repair pathway by recognizing specific bases in DNAand cleaving the sugar base bond to release the damaged base (David etal., Chem. Rev. 98: 1221-1262, 1998).

The Neil genes are a newly discovered family of mammalian DNAN-glycosylases related to the Fpg/Nei family of proteins fromEscherichia coli (Hazra et al., Proc. Natl. Acad. Sci. USA 99:3523-3528, 2002; Bandaru et al., DNA Repair 1: 517-529, 2002). Neil1recognizes and removes a wide spectrum of oxidized pyrimidines andring-opened purines from DNA, including thymine glycol (Tg),2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and4,6-diamino-5-formidopyrimidine (FapyA). Tg, FapyG and FapyA are amongthe most prevalent oxidized bases produced by ionizing radiation(Dizdaroglu et al. Free Radic. Biol. Med. 32: 1102-1115, 2002) and canblock replicative DNA polymerases, which can, in turn, cause cell death(Asagoshi et al. J. Biol. Chem. 277: 14589-14597, 2002; Clark et al.,Biochemistry 28: 775-779, 1989).

The Nth1 and Ogg1 glycosylases each remove subsets of oxidized DNA basesthat overlap with substrates of Neil1 (Nishimura, Free Radic. Biol. Med.32: 813-821, 2002; Asagoshi et al., Biochemistry 39: 11389-11398, 2000;Dizdaroglu et al., Biochemistry 38: 243-246, 1999). However, mice withnull mutations in either Nth1 (Ocampo et al., Mol. Cell. Biol. 22:6111-6121, 2002; Takao et al., EMBO J. 21: 3486-3493, 2002) or Ogg1(Klungland et al., Proc. Natl. Acad. Sci. USA 96: 13300-13305, 1999;Minowa et al., Proc. Natl. Acad. Sci. USA 97: 4156-4161, 2000) areviable, raising the possibility that Neil1 activity tempers the loss ofNth1 or Ogg1. Recently, a residual Tg-DNA glycosylase activity inNth1^(−/−) mice has been identified as Neil1 (Takao et al., J. Biol.Chem. 277: 42205-42213, 2002).

We constructed a single shRNA expression vector targeting a sequencenear the 5′ end of the Neil1 coding region. This vector was introducedinto mouse embryonic stem cells by electroporation, and individualstable integrants were tested for expression of the Neil1 protein (seethe weblink: http://www.cshl.edu/public/SCIENCE/hannon.html for detailedprocedures). The majority of cell lines showed an ˜80% reduction inNeil1 protein, which correlated with a similar change in levels of Neil1mRNA. These cells showed an approximately two-fold increase in theirsensitivity to ionizing radiation, consistent with a role for Neil1 inDNA repair. Two independent ES cell lines were injected into BL/6blastocysts, and several high-percentage chimeras were obtained. Thesechimeras were out-crossed, and germ-line transmission of theshRNA-expression construct was noted in numerous F₁ progeny (13/27 forone line and 12/26 for the other).

To determine whether the silencing of Neil1 that had been observed in EScells was transmitted faithfully, we examined Neil1 mRNA and proteinlevels. Both were reduced by approximately the same extent that had beenobserved in the engineered ES cells (FIGS. 55, 56). Consistent with thishaving occurred through the RNAi pathway, we detected the presence ofsiRNAs corresponding to the shRNA sequence in F₁ animals that carry theshRNA expression vector but not in those that lack the vector (FIG. 56b).

The aforementioned data demonstrate that shRNAs can be used to creategerm-line transgenic mice in which RNAi has silenced a target gene.These observations open the door to using of RNAi as a complement tostandard knock-out methodologies and provide a means to rapidly assessthe consequences of suppressing a gene of interest in a living animal.Coupled with activator-dependent U6 promoters, the use of shRNAs willultimately provide methods for tissue-specific, inducible and reversiblesuppression of gene expression in mice.

EXAMPLE 12 Dicer Cleaves a Single siRNA from the End of Each shRNA

We performed the following experiments in order to understand how Dicerprocesses shRNAs, and in order to permit comparison of the efficiency ofdifferent silencing triggers.

We began by producing ˜70 chemically synthesized shRNAs, targetingvarious endogenous genes and reporters. We initially focused on adetailed analysis of one set of four shRNAs that target fireflyluciferase (FIG. 57 a). The individual species differed in two distinctways. First, the stems of the shRNAs were either 19 or 29 nucleotides inlength. Second, each shRNA either contained or lacked a 2 nucleotide 3′overhang, identical to that produced by processing of pri-miRNAs byDrosha. Each species was end-labeled by enzymatic phosphorylation andincubated with recombinant human Dicer. The 29 nt. shRNA bearing the 3′overhang was converted almost quantitatively into a 22 nt product byDicer (FIG. 57 b). In contrast, the 29 nt shRNA that lacked the overhanggenerated very little 22 nt labeled product, although there was asubstantial depletion of the starting material. Neither 19 nt shRNA wascleaved to a significant extent by the Dicer enzyme. This result was notdue to the lack of dsRNA in the 19 nt shRNAs as all shRNA substrateswere efficiently cleaved by bacterial RNAseIII (FIG. 57 c). Parallelanalysis of identical shRNA substrates that were produced by in vitrotranscription with T7 polymerase and uniformly labeled clarified theresults obtained with end-labeled substrates (not shown). Specifically,19 nt shRNAs were not cleaved. However, both the overhung and the blunt29 nucleotide shRNAs gave rise to 22 nt products, albeit at reducedlevels in the latter case. These results suggest that Dicer requires aminimum stem length for productive cleavage. Furthermore, they areconsistent with a hypothesis that the presence of a correct 3′ overhangenhances the efficiency and specificity of cleavage, directing Dicer tocut ˜22 nucleotides from the end of the substrate.

A number of previous studies have suggested that Dicer might function asan end-recognizing endonuclease, without positing a role for the 3′overhang. Processive Dicer cleavage was first implied by in vitroanalysis of RISC cleavage (Zamore et al., Cell 101: 25-33, 2000). InDrosophila embryo extracts programmed for RISC assembly using a longdsRNA, phased cleavage sites occurred at approximately 22 nucleotideintervals along an mRNA substrate. Similarly, analysis of C. elegansDicer in whole cell extracts (Ketting et al., Genes Dev 15: 2654-9,2001) or purified human Dicer in vitro (Zhang et al., EMBO J 21:5875-85, 2002) showed accumulation of discretely sized cleavageintermediates. Blocking of the ends of dsRNAs using either fold-backstructures or chimeric RNA-DNA hybrids attenuated, but did not abolish,the ability of human Dicer to generate siRNAs (Zhang et al., EMBO J 21:5875-85, 2002). Finally, Lund and colleagues suggested that Dicercleaved ˜22 nt from the blunt end of an extended pre-miRNA, designed inpart to mimic a pri-miRNA (see Lund et al., Science 303: 95-8, 2004).

Our results suggest that while the overhang is not obligate for Dicerprocessing of its substrates (see Zhang et al., EMBO J 21: 5875-85,2002, and FIG. 57 b), this structure does aid in determining thespecificity of cleavage. Furthermore, time courses of processing ofblunt and overhung 29 nt shRNAs do show a more rapid processing of theoverhung substrate if reactions are performed in the linear range forthe enzyme (not shown).

To map more precisely the position of Dicer cleavage in the shRNA, weused primer extension analysis. The shRNAs described in FIG. 57 a werereacted with recombinant human Dicer as shown in FIG. 57 b. Total RNAwas recovered from the processing reactions and used in primer extensionassays. Consistent with direct analysis of the RNA, shRNAs with 19 ntstems failed to yield discrete extension products. The extensionproducts that would be predicted from the unreacted substrate are notseen due to secondary structure of the uncleaved precursor (FIG. 58 a).Both of the 29 nt shRNAs give rise to extension products with theoverhung precursor giving a relatively discrete product of 20nucleotides, as predicted for a cleavage precisely 22 nt from the 3′ endof the substrate (FIG. 58 b). The blunt-ended precursor gave adistribution of products, as was predicted from the analysis ofuniformly and end-labeled RNAs.

In Drosophila, Dicer2 acts in a complex with a double-stranded RNAbinding protein, R2D2 (Liu et al., Science 301: 1921-5, 2003).Similarly, biochemical evidence from C. elegans suggests that its Dicerbinds RDE-1, RDE-4 and DRH-1 (Tabara et al., Cell 109: 861-71, 2002).These results suggest that the human enzyme might also function as partof a larger complex, which could show altered cleavage specificities.Therefore, we also mapped the cleavage of our shRNAs in vitro.Precursors were transfected into cells, and the processed form of eachwas isolated by virtue of its co-immunoprecipitation with humanArgonaute proteins, Ago1 and Ago2. Primer extension suggested identicalcleavage specificities upon exposure of shRNAs to Dicer in vitro and inliving cells (FIG. 58 c).

EXAMPLE 13 shRNAs are Generally more Effective than siRNAs

Since each shRNA gave rise to a single, predictable 22 nt sequence inRISC, we compared the efficacy of shRNAs and siRNAs. Toward this goal,we selected 43 sequences targeting a total of 6 genes (3-9 sequences pergene). For each sequence, we synthesized a 21 nt siRNA (19 base stem)and 19 and 29 nt shRNAs that were predicted to give Dicer products thatwere either identical to the siRNAs or that differed by the addition ofone 3′ nucleotide (FIG. 59 a). Each RNA species was transfected intoHeLa cells at a relatively high concentration (100 nM). The level ofsuppression was determined by semi-quantitative RT-PCR and theperformance of each shRNA compared to the performance of thecorresponding siRNA (FIG. 59 b). Comparison of 19 nt shRNAs with siRNAsrevealed that there was little difference in endpoint inhibition withthese species (left panel). A comparison of siRNAs with 29 nt shRNAsgave a different result. Clustering of the comparison data points abovethe diagonal indicated consistently better endpoint inhibition with the29 nt shRNAs (right panel).

The generally better endpoint inhibition observed with 29 nt shRNAs ledus to investigate in more detail the performance of these silencingtriggers as compared to siRNAs. Seventeen complete sets comprising ansiRNA, a 19 nt shRNA and a 29 nt shRNA were examined for suppression intitration experiments. In all cases, the 19 nt shRNAs performed as wellas or worse than the corresponding siRNAs. In contrast, 29 nt shRNAsexceeded the performance of siRNAs in the majority of cases. Fourrepresentative examples, targeting MAPK-14 are shown in FIG. 59 c.Several 29 nt shRNAs (e.g., see MAPK14-1) showed both significantlygreater endpoint inhibition and efficacy at lower concentrations thanthe corresponding siRNA. In other cases( e.g., see MAPK14-2 andMAPK-14-4), the maximal level of suppression for the 29 nt. shRNA wasapproximately two-fold greater than the maximal level of suppression forthe corresponding siRNA. Finally, in a minority of cases, exemplified byMAPK14-3, the performance of the three types of silencing triggers wassimilar. Importantly, in only one case out of 17 did we note that the 29nt shRNA with a 2 nt. 3′ overhang performed less effectively than thecorresponding siRNA (data not shown).

EXAMPLE 14 siRNAs and shRNAs give Similar Profiles of Off-Target Effectsat Saturation

Sequence specificity is a critical parameter in RNAi experiments.Microarray analysis has revealed down-regulation of many non-targetedtranscripts following transfection of siRNAs into HeLa cells (Jackson etal., Nat Biotechnol 21: 635-7, 2003). Notably, these gene expressionsignatures differed between different siRNAs targeting the same gene.Many of the “off target” transcripts contained sites of partial identityto the individual siRNA, possibly explaining the source of the effects.To examine potential off-target effects of synthetic shRNAs, we comparedshRNA signatures with those of siRNAs derived from the same targetsequence. Using microarray gene expression profiling, we obtained agenome-wide view of transcript suppression in response to siRNA andshRNA transfection. FIG. 60(a and b) shows heat maps of signaturesproduced in HeLa cells 24 hours after transfection of 19 nt and 29 ntshRNAs compared with those generated by corresponding siRNAs. 19 ntshRNAs produced signatures that resembled, but were not identical to,those of corresponding siRNAs. In contrast, the signatures of the 29 ntshRNAs (FIG. 60 a) were nearly identical to those of the siRNAs.

These results indicate that off target effects may be inherent to theuse of synthetic RNAs for eliciting RNAi and cannot be ameliorated byintracellular processing of an upstream precursor in the RNAi pathway.Furthermore, the agreement between the signatures of 29 nt shRNAs andsiRNAs is consistent with precise intracellular processing of the shRNAto generate a single siRNA rather than a random sampling of the hairpinstem by Dicer. The basis of the divergence between the signature of the19 nt shRNA and the corresponding siRNA is presently unclear.

Considered together, our results indicate that chemically synthesized,29 nt shRNAs are often substantially more effective triggers of RNAithan are siRNAs. While not wishing to be bound by any particular theory,a possible mechanistic explanation for this finding may lie in the factthat 29 nt shRNAs are substrates for Dicer processing both in vitro andin vitro. We originally suggested that siRNAs might be passed from Dicerto RISC in a solid state reaction on the basis of an interaction betweenDicer and Argonaute2 in Drosophila S2 cell extracts (Hammond et al.,Science 293: 1146-50, 2001). More recently, results from severallaboratories have strongly suggested a model for assembly of the RNAieffector complex in which a multi-protein assembly containing Dicer andaccessory proteins interacts with an Argonaute protein and activelyloads one strand of the siRNA or miRNA into RISC (Lee et al., Cell 117:69-81, 2004; Pham et al., Cell 117: 83-94, 2004; Tomari et al., Cell116: 831-41, 2004). Our result is consistent with a model where Dicersubstrates, derived from nuclear processing of pri-miRNAs or cytoplasmicdelivery of pre-miRNA mimetics, are loaded into RISC more effectivelythan siRNAs. Our data support such a model, since it is not the hairpinstructure of the synthetic RNA that determines its increased efficacybut the fact that the shRNA is a Dicer substrate that correlates withenhanced potency. Again, not wishing to be bound by any particulartheory, it is possible that even siRNAs enter RISC via a Dicer-mediatedassembly pathway. Our data may also reflect an increased affinity ofDicer for longer duplexes substrates. Alternatively, hairpin RNAs, suchas miRNA precursors, might interact with specific cellular proteins thatfacilitate delivery of these substrates to Dicer, whereas siRNAs mightnot benefit from such chaperones.

Overall, our results provide an improved method for triggering RNAi inmammalian cells that uses higher potency RNAi triggers. Mapping thesingle 22 nt sequence that appears in RISC from each of these shRNAs nowpermits the combination of this more effective triggering method withrules for effective siRNA design.

Methods

RNA Sequence Design

Each set of RNAs began with the choice of a single 19-mer sequence.These 19 mers were used directly to create siRNAs. To create shRNAs with19-mer stems, we appended a 4-base loop (either CCAA or UUGG) to the endof the 19-mer sense strand target sequence followed by the 19-mercomplementary sequence and a UU overhang. To create 29-mer stems, weincreased the length of the 19-mer target sequence by adding 1 baseupstream and 9 bases downstream from the target region and used the sameloop sequence and UU overhang. All synthetic RNA molecules used in thisstudy were purchased from Dharmacon.

Dicer Processing

RNA hairpins corresponding to luciferase were end-labeled with [γ-³²P]ATP and T4 Polynucleotide kinase. 0.1 pmoles of RNA were then processedwith 2 units of Dicer (Stratagene) at 37° C. for 2 hours. Reactionproducts were trizol extracted, isopropanol precipitated, run on an 18%polyacrylamide, 8M urea denaturing gel. For RNaseIII digestion, 0.1pmoles were digested with 1 unit of E. coli RNase III (NEB) for 30minutes at 37° C. and analyzed as described above. For primer extensionanalysis, hairpins were processed with Dicer at 37° C. for 2 hours,followed by heat inactivation of the enzyme. DNA primers were 5′ labeledwith PNK and annealed to 0.05 pmole of RNA as follows: 95° C. for oneminute, 10 minutes at 50° C. and then 1 min on ice. Extensions werecarried out at 42° C. for 1 hour using MoMLV reverse transcriptase.Products were analyzed by electrophoresis on a 8M Urea/20%polyacrylamide gel. For analysis of in vitro processing, LinxA cellswere transfected in 10 cm plates using Mirus TKO (10 μg hairpin RNA) orMirus LT4 reagent for DNA transfection (12 μg of tagged Ago1/Ago 2 DNA;J. Liu, unpublished). Cells were lysed and immunoprecipitated after 48hours using with myc Antibody (9E14) Antibody. Immuno-precipitationswere washed 3× in lysis buffer and treated with DNase for 15 minutes.Immunoprecipitates were then primer extended as described above.

siRNA and shRNA Transfections and mRNA Quantitation

HeLa cells were transfected in 96-well plates by use of Oligofectamine(Invitrogen) with the final nanomolar concentrations of each syntheticRNA indicated in the graphs. RNA quantitation was performed by Real-timePCR, using appropriate Applied Biosystems TaqMan™ primer probe sets. Theprimer probe set used for MAPK14 was Hs00176247_m1. RNA values werenormalized to RNA for HGUS (probe 4310888E).

Microarray Gene Expression Profiling

HeLa cells were transfected in 6-well plates by use of Oligofectamine.RNA from transfected cells was hybridized competitively with RNA frommock-transfected cells (treated with transfection reagent in the absenceof synthetic RNA). Total RNA was purified by Qiagen RNeasy kit, andprocessed as described previously (Hughes et al., Nat Biotechnol 19:342-7, 2001) for hybridization to microarrays containingoligonucleotides corresponding to approximately 21,000 human genes.Ratio hybridizations were performed with fluorescent label reversal toeliminate dye bias. Microarrays were purchased from AgilentTechnologies. Error models have been described previously (Hughes etal., Nat Biotechnol 19: 342-7, 2001). Data were analyzed using RosettaResolver™ software. SUPPLEMENTARY TABLE 1 Sequences of the siRNAs usedin this study Accession Target sequence Gene number ID Target sequenceIGF1R NM_000875 IGF1R-1 GGAUGCACCAUCUUCAAGG (SEQ ID NO: 47) IGF1RNM_000875 IGF1R-2 GACAAAAUCCCCAUCAGGA (SEQ ID NO: 48) IGF1R NM_000875IGF1R-3 ACCGCAAAGUCUUUGAGAA (SEQ ID NO: 49) IGF1R NM_000875 IGF1R-4GUCCUGACAUGCUGUUUGA (SEQ ID NO: 50) IGF1R NM_000875 IGFlR-5GACCACCAUCAACAAUGAG (SEQ ID NO: 51) IGFlR NM_000875 IGF1R-6CAAAUUAUGUGUUUCCGAA (SEQ ID NO: 52) IGF1R NM_000875 IGF1R-7CGCAUGUGCUGGCAGUAUA (SEQ ID NO: 53) IGF1R NM_000875 IGF1R-8CCGAAGAUUUCACAGUCAA (SEQ ID NO: 54) IGF1R NM_000875 IGF1R-9ACCAUUGAUUCUGUUACUU (SEQ ID NO: 55) KIF11 NM_004523 KIF11-1CUGACAAGAGCUCAAGGAA (SEQ ID NO: 56) KIF11 NM_004523 KIF11-2CGUUCUGGAGCUGUUGAUA (SEQ ID NO: 57) KIF11 NM_004523 KIF11-3GAGCCCAGAUCAACCUUUA (SEQ ID NO: 58) KIF11 NM_004523 KIF11-4GGCAUUAACACACUGGAGA (SEQ ID NO: 59) KIF11 NM_004523 KIF11-5GAUGGCAGCUCAAAGCAAA (SEQ ID NO: 60) KIF11 NM_004523 K1F11-6CAGCAGAAAUCUAAGGAUA (SEQ ID NO: 61) KIF14 NM_014875 KIF14-1CAGGGAUGCUGUUUGGAUA (SEQ ID NO: 62) KIF14 NM_014875 KIF14-2ACUGACAACAAAGUGCAGC (SEQ ID NO: 63) KIF14 NM_014875 KIF14-3AAACUGGGAGGCUACUUAC (SEQ ID NO: 64) KIF14 NM_014875 KIF14-4CACUGAAUGUGGGAGGUGA (SEQ ID NO: 65) KIF14 NM_014875 K1F14-5GUCUGGGUGGAAAUUCAAA (SEQ ID NO: 66) KIF14 NM_014875 KIF14-6CAUCUUUGCUGAAUCGAAA (SEQ ID NO: 67) KIF14 NM_014875 K1F14-7GGGAUUGACGGCAGUAAGA (SEQ ID NO: 68) KIF14 NM_014875 KIF14-8CAGGUAAAGUCAGAGACAU (SEQ ID NO: 69) KIF14 NM_014875 KIF14-9CUCACAUUGUCCACCAGGA (SEQ ID NO: 70) KNSL1 NM_004523 KNSL1-1GACCUGUGCCUUUUAGAGA (SEQ ID NO: 71) KNSL1 NM_004523 KNSL1-2AAAGGACAACUGCAGCUAC (SEQ ID NO: 72) KNSL1 NM_004523 KNSL1-3GACUUCAUUGACAGUGGCC (SEQ ID NO: 73) MAPK14 NM 139012 MAPK14-1AAUAUCCUCAGGGGUGGAG (SEQ ID NO: 74) MAPK14 NM_139012 MAPK14-2GUGCCUCUUGUUGCAGAGA (SEQ ID NO: 75) MAPK14 NM_139012 MAPK14-3GAAGCUCUCCAGACCAUUU (SEQ ID NO: 76) MAPK14 NM_001315 MAPK14-4CUCCUGAGAUCAUGCUGAA (SEQ ID NO: 77) MAPK14 NM 001315 MAPK14-5GCUGUUGACUGGAAGAACA (SEQ ID NO: 78) MAPK14 NM 001315 MAPK14-6GGAAUUCAAUGAUGUGUAU (SEQ ID NO: 79) MAPK14 NM_001315 MAPK14-7CCAUUUCAGUCCAUCAUUC (SEQ ID NO: 80) PLK NM_005030 PLK-1CCCUGUGUGGGACUCCUAA (SEQ ID NO: 81) PLK NM_005030 PLK-2CCGAGUUAUUCAUCGAGAC (SEQ ID NO: 82) PLK NM_005030 PLK-3GUUCUUUACUUCUGGCUAU (SEQ ID NO: 83) PLK NM_005030 PLK-4CGCCUCAUCCUCUACAAUG (SEQ ID NO: 84) PLK NM_005030 PLK-5AAGAGACCUACCUCCGGAU (SEQ ID NO: 85) PLK NM_005030 PLK-6GGUGUUCGCGGGCAAGAUU (SEQ ID NO: 86) PLK NM_005030 PLK-7CUCCUUAAAUAUUUCCGCA (SEQ ID NO: 87) PLK NM_005030 PLK-8AAGAAGAACCAGUGGUUCG (SEQ ID NO: 88) PLK NM_005030 PLK-9CUGAGCCUGAGGCCCGAUA (SEQ ID NO: 89)Literature Cited

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V. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. All of theabove-cited references and publications are hereby incorporated byreference.

1. A method for attenuating expression of a target gene in mammaliancells, comprising introducing into the mammalian cells a single-strandedhairpin ribonucleic acid (shRNA) comprising self complementary sequencesof 19 to 100 nucleotides that form a duplex region, which selfcomplementary sequences hybridize under intracellular conditions to atarget gene, wherein said hairpin RNA: (i) is a substrate for cleavageby a RNaseIII enzyme to produce a double-stranded RNA product, (ii) doesnot produce a general sequence-independent killing of the mammaliancells, and (iii) reduces expression of said target gene in a mannerdependent on the sequence of said complementary regions, and, whereinsaid shRNA comprises a 3′ overhang of about 1-4 nucleotides.
 2. A methodfor attenuating expression of a target gene in mammalian cells,comprising introducing into the mammalian cells a single-strandedhairpin ribonucleic acid (shRNA) comprising self complementary sequencesof 19 to 100 nucleotides that form a duplex region, which selfcomplementary sequences hybridize under intracellular conditions to atarget gene, wherein said hairpin RNA: (i) is cleaved in the mammaliancells to produce an RNA guide sequence that enters anArgonaut-containing complex, (ii) does not produce a generalsequence-independent killing of the mammalian cells, and (iii) reducesexpression of said target gene in a manner dependent on the sequence ofsaid complementary regions, and, wherein said shRNA comprises a 3′overhang of about 1-4 nucleotides.
 3. A method for attenuatingexpression of one or more target genes in mammalian cells, comprisingintroducing into the mammalian cells a variegated library of single-stranded hairpin ribonucleic acid (shRNA) species, each shRNA speciescomprising self complementary sequences of 19 to 100 nucleotides thatform duplex regions and which hybridize under intracellular conditionsto a target gene, wherein each of said hairpin RNA species: (i) is asubstrate for cleavage by a RNaseIII enzyme to produce a double-strandedRNA product, (ii) does not produce a general sequence-independentkilling of the mammalian cells, and (iii) if complementary to a targetsequence, reduces expression of said target gene in a manner dependenton the sequence of said complementary regions, and, wherein said shRNAcomprises a 3′ overhang of about 1-4 nucleotides.
 4. The method of claim1, 2, or 3, wherein the shRNA comprises a 3′ overhang of 2 nucleotides.5. The method of claim 1, 2, or 3, wherein the shRNA comprisesself-complementary sequences of 25 to 29 nucleotides that form duplexregions.
 6. The method of claim 1, 2, or 3, wherein theself-complementary sequences are 29 nucleotides in length.
 7. The methodof claim 1, 2, or 3, wherein the shRNA is transfected or microinjectedinto said mammalian cells.
 8. The method of claim 1, 2, or 3, whereinthe shRNA is a transcriptional product that is transcribed from anexpression construct introduced into said mammalian cells, whichexpression construct comprises a coding sequence for transcribing saidshRNA, operably linked to one or more transcriptional regulatorysequences.
 9. The method of claim 8, wherein said transcriptionalregulatory sequences include a promoter for an RNA polymerase.
 10. Themethod of claim 9, wherein said RNA polymerase is a cellular RNApolymerase.
 11. The method of claim 9, wherein said promoter is a U6promoter, a T7 promoter, a T3 promoter, or an SP6 promoter.
 12. Themethod of claim 8, wherein said transcriptional regulatory sequencesincludes an inducible promoter.
 13. The method of claim 8, wherein saidmammalian cells are stably transfected with said expression construct.14. The method of claim 1, 2 or 3, wherein the mammalian cells areprimate cells.
 15. The method of claim 14, wherein the primate cells arehuman cells.
 16. The method of claim 1 or 2, wherein the shRNA isintroduced into the mammalian cells in cell culture or in an animal. 17.The method of claim 1 or 2, wherein expression of the target isattenuated by at least 33 percent relative expression in cells nottreated said hairpin RNA.
 18. The method of claim 1 or 2, wherein thetarget gene is an endogenous gene or a heterologous gene relative to thegenome of the mammalian cell.
 19. The method of claim 1 or 2, whereinthe self complementary sequences hybridize under intracellularconditions to a non-coding sequence of the target gene selected from apromoter sequence, an enhancer sequence, or an intronic sequence. 20.The method of claim 1 or 2, wherein the shRNA includes one or moremodifications to phosphate-sugar backbone or nucleosides residues. 21.The method of claim 3, wherein said variegated library of shRNA speciesare arrayed a solid substrate.
 22. The method of claim 3, including thefurther step of identifying shRNA species of said variegated librarywhich produce a detected phenotype in said mammalian cells.
 23. Themethod of claim 1, 2, or 3, wherein the shRNA is a chemicallysynthesized product or an in vitro transcription product.
 24. A methodof enhancing the potency/activity of an RNAi therapeutic for a mammalianpatient, said RNAi therapeutic comprising an siRNA of 19-22 pairedpolynucleotides, the method comprising replacing said siRNA with asingle-stranded hairpin RNA (shRNA) of claim 1 or 2, wherein said duplexregion comprises the same 19-22 paired polynucleotides of said siRNA.25. The method of claim 24, wherein said shRNA comprises a 3′ overhangof 2 nucleotides.
 26. The method of claim 24, wherein the half-maximuminhibition by said RNAi therapeutic is achieved by a concentration ofsaid shRNA at least about 20% lower than that of said siRNA.
 27. Themethod of claim 26, wherein the half-maximum inhibition by said RNAitherapeutic is achieved by a concentration of said shRNA at least about100% lower than that of said siRNA.
 28. The method of claim 24, whereinthe end-point inhibition by said shRNA is at least about 40% higher thanthat of said siRNA.
 29. The method of claim 24, wherein the end-pointinhibition by said shRNA is at least about 2-6 fold higher than that ofsaid siRNA.
 30. A method of designing a short hairpin RNA (shRNA)construct for RNAi, said shRNA comprising a 3′ overhang of about 1-4nucleotides, the method comprising selecting the nucleotide about 21bases 5′ to the most 3′-end nucleotide as the first paired nucleotide ina cognate doubled-stranded siRNA with the same 3′ overhang.
 31. Themethod of claim 30, wherein said shRNA comprises 25-29 pairedpolynucleotides.
 32. The method of claim 31, wherein said shRNA, whencut by a Dicer enzyme, produces a product siRNA that is either identicalto, or differ by a single basepair immediately 5′ to the 3′ overhangfrom, said cognate siRNA.
 33. The method of claim 32, wherein said Dicerenzyme is a human Dicer.
 34. The method of claim 30, wherein said 3′overhang has 2 nucleotides.
 35. The method of claim 30, wherein saidshRNA is for RNAi in mammalian cells.